bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Cardiac Efficiency and Altered Substrate Metabolism Precedes the Onset of Hyperglycemia and Contractile Dysfunction in Two Mouse Models of Insulin Resistance and Obesity
Authors: Buchanan Jonathan , Mazumder Pradip K. , Hu Ping , Chakrabarti Gopa , Roberts Matthew W. , Yun Ui Jeong , Cooksey Robert C. , Litwin Sheldon E. , Abel E. Dale
Published: September 2005

Hyperglycemia is associated with altered myocardial substrate use, a condition that has been hypothesized to contribute to impaired cardiac performance. The goals of this study were to determine whether changes in cardiac metabolism, gene expression, and function precede or follow the onset of hyperglycemia in two mouse models of obesity, insulin resistance, and diabetes (ob/ob and db/db mice). Ob/ob and db/db mice were studied at 4, 8, and 15 wk of age. Four-week-old mice of both strains were normoglycemic but hyperinsulinemic. Hyperglycemia develops in db/db mice between 4 and 8 wk of age and in ob/ob mice between 8 and 15 wk. In isolated working hearts, rates of glucose oxidation were reduced by 28–37% at 4 wk and declined no further at 15 wk in both strains. Fatty acid oxidation rates and myocardial oxygen consumption were increased in 4-wk-old mice of both strains. Fatty acid oxidation rates progressively increased in db/db mice in parallel with the earlier onset and greater duration of hyperglycemia. In vivo, cardiac catheterization revealed significantly increased left ventricular contractility and relaxation (positive and negative dP/dt) in both strains at 4 wk of age. dP/dt declined over time in db/db mice but remained elevated in ob/ob mice at 15 wk of age. Increased β-myosin heavy chain isoform expression was present in 4-wk-old mice and persisted in 15-wk-old mice. Increased expression of peroxisomal proliferator-activated receptor-α regulated genes was observed only at 15 wk in both strains. These data indicate that altered myocardial substrate use and reduced myocardial efficiency are early abnormalities in the hearts of obese mice and precede the onset of hyperglycemia. Obesity per se does not cause contractile dysfunction in vivo, but loss of the hypercontractile phenotype of obesity and up-regulation of peroxisomal proliferator-activated receptor-α regulated genes occur later and are most pronounced in the presence of longstanding hyperglycemia.
Hypothalamic 3′,5′-Cyclic Adenosine Monophosphate Response Element-Binding Protein Loss Causes Anterior Pituitary Hypoplasia and Dwarfism in Mice
Authors: Mantamadiotis Theo , Kretz Oliver , Ridder Stephanie , Bleckmann Susanne C. , Bock Dagmar , Gröne Hermann-Josef , Malaterre Jordane , Dworkin Sebastian , Ramsay Robert G. , Schütz Günther
Published: September 2005

The principal regulation of body growth is via a cascade of hormone signals emanating from the hypothalamus, by release of GHRH, which then directs the somatotroph cells of the pituitary to release GH into the blood stream. This in turn leads to activation of signal transducer and activator of transcription 5-dependent expression of genes such as IGF-I in hepatocytes, acid labile substance, and serine protease inhibitor 2.1, resulting in body growth. Here, using conditional cAMP response element binding protein (CREB) mutant mice, we show that loss of the CREB transcription factor in the brain, but not the pituitary, results in reduced postnatal growth consistent with dwarfism caused by GH deficiency. We demonstrate that although there appears to be no significant impact upon the expression of GHRH mRNA in CREB mutant mice, the amount of GHRH peptide is reduced. These findings show that CREB is required for the efficient production of GHRH in hypothalamus, in addition to its previously reported role in pituitary GH production and somatotroph expansion.
The Notch ligands DLL1 and JAG2 act synergistically to regulate hair cell development in the mammalian inner ear
Authors: Kiernan Amy E. , Cordes Ralf , Kopan Raphael , Gossler Achim , Gridley Thomas
Published: September 2005

The mammalian auditory sensory epithelium, the organ of Corti, contains sensory hair cells and nonsensory supporting cells arranged in a highly patterned mosaic. Notch-mediated lateral inhibition is the proposed mechanism for creating this sensory mosaic. Previous work has shown that mice lacking the Notch ligand JAG2 differentiate supernumerary hair cells in the cochlea,consistent with the lateral inhibitory model. However, it was not clear why only relatively modest increases in hair cell production were observed in Jag2 mutant mice. Here, we show that another Notch ligand, DLL1,functions synergistically with JAG2 in regulating hair cell differentiation in the cochlea. We also show by conditional inactivation that these ligands probably signal through the NOTCH1 receptor. Supernumerary hair cells in Dll1/Jag2 double mutants arise primarily through a switch in cell fate, rather than through excess proliferation. Although these results demonstrate an important role for Notch-mediated lateral inhibition during cochlear hair cell patterning, we also detected abnormally prolonged cellular proliferation that preferentially affected supporting cells in the organ of Corti. Our results demonstrate that the Notch pathway plays a dual role in regulating cellular differentiation and patterning in the cochlea, acting both through lateral inhibition and the control of cellular proliferation.
Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture
Authors: Tada Shinsuke , Era Takumi , Furusawa Chikara , Sakurai Hidetoshi , Nishikawa Satomi , Kinoshita Masaki , Nakao Kazuki , Chiba Tsutomu , Nishikawa Shin-Ichi
Published: September 2005

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+E-cadherin(ECD)+PDGFRα(αR)+population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ECD+αR- and Gsc+ECD-αR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages,respectively. The presence of mesendodermal cells in nascent Gsc+ECD+αR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.
Specification and maintenance of the spinal cord stem zone
Authors: Delfino-Machín Mariana , Lunn J. Simon , Breitkreuz Dorette N. , Akai Jun , Storey Kate G.
Published: September 2005

Epiblast cells adjacent to the regressing primitive streak behave as a stem zone that progressively generates the entire spinal cord and also contributes to paraxial mesoderm. Despite this fundamental task, this cell population is poorly characterised, and the tissue interactions and signalling pathways that specify this unique region are unknown. Fibroblast growth factor (FGF) is implicated but it is unclear whether it is sufficient and/or directly required for stem zone specification. It is also not understood how establishment of the stem zone relates to the acquisition of spinal cord identity as indicated by expression of caudal Hox genes.

Here, we show that many cells in the chick stem zone express both early neural and mesodermal genes; however, stem zone-specific gene expression can be induced by signals from underlying paraxial mesoderm without concomitant induction of an ambivalent neural/mesodermal cell state. The stem zone is a site of FGF/MAPK signalling and we show that although FGF alone does not mimic paraxial mesoderm signals, it is directly required in epiblast cells for stem zone specification and maintenance. We further demonstrate that caudal Hox gene expression in the stem zone also depends on FGF and that neither stem zone specification nor caudal Hox gene onset requires retinoid signalling. These findings thus support a two step model for spinal cord generation -FGF-dependent establishment of the stem zone in which progressively more caudal Hox genes are expressed, followed by the retinoid-dependent assignment of spinal cord identity.
Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate
Authors: Yu Ling , Gu Shuping , Alappat Sylvia , Song Yiqiang , Yan Mingquan , Zhang Xiaoyun , Zhang Guozhong , Jiang Yiping , Zhang Zunyi , Zhang Yanding , Chen YiPing
Published: September 2005

The short stature homeobox gene SHOX is associated with idiopathic short stature in humans, as seen in Turner syndrome and Leri-Weill dyschondrosteosis, while little is known about its close relative SHOX2. We report the restricted expression of Shox2 in the anterior domain of the secondary palate in mice and humans. Shox2-/- mice develop an incomplete cleft that is confined to the anterior region of the palate, an extremely rare type of clefting in humans. The Shox2-/- palatal shelves initiate, grow and elevate normally, but the anterior region fails to contact and fuse at the midline, owing to altered cell proliferation and apoptosis, leading to incomplete clefting within the presumptive hard palate. Accompanied with these cellular alterations is an ectopic expression of Fgf10 and Fgfr2c in the anterior palatal mesenchyme of the mutants. Tissue recombination and bead implantation experiments revealed that signals from the anterior palatal epithelium are responsible for the restricted mesenchymal Shox2 expression. BMP activity is necessary but not sufficient for the induction of palatal Shox2 expression. Our results demonstrate an intrinsic requirement for Shox2 in palatogenesis, and support the idea that palatogenesis is differentially regulated along the anteroposterior axis. Furthermore, our results demonstrate that fusion of the posterior palate can occur independently of fusion in the anterior palate.
Toutatis, a TIP5-related protein, positively regulates Pannier function during Drosophila neural development
Authors: Vanolst Luc , Fromental-Ramain Catherine , Ramain Philippe
Published: September 2005

The GATA factor Pannier (Pnr) activates proneural expression through binding to a remote enhancer of the achaete-scute (ac-sc)complex. Chip associates both with Pnr and with the (Ac-Sc)-Daughterless heterodimer bound to the ac-sc promoters to give a proneural complex that facilitates enhancer-promoter communication during development. Using a yeast two-hybrid screening, we have identified Toutatis (Tou), which physically interacts with both Pnr and Chip. Loss-of-function and gain-of-function experiments indicate that Tou cooperates with Pnr and Chip during neural development. Tou shares functional domains with chromatin remodelling proteins, including TIP5 (termination factor TTFI-interacting protein 5) of NoRC (nucleolar remodelling complex), which mediates repression of RNA polymerase 1 transcription. In contrast, Tou acts positively to activate proneural gene expression. Moreover, we show that Iswi associates with Tou, Pnr and Chip, and is also required during Pnr-driven neural development. The results suggest that Tou and Iswi may belong to a complex that directly regulates the activity of Pnr and Chip during enhancer-promoter communication, possibly through chromatin remodelling.
Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation inDrosophilafollicle cells
Authors: Sun Jianjun , Deng Wu-Min
Published: September 2005

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. We show that the homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut was expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression was controlled by the Notch pathway and was essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells entered the endocycle and differentiated prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut caused defects in the mitotic cycle/endocycle switch. These cells continued to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promoted Cyclin A expression by negatively regulating Fizzy-related. Our data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells.
Agrin regulates growth cone turning ofXenopusspinal motoneurons
Authors: Xu Xiaohua , Fu Amy K. Y. , Ip Fanny C. F. , Wu Chien-ping , Duan Shumin , Poo Mu-ming , Yuan Xiao-bing , Ip Nancy Y.
Published: September 2005

The pivotal role of agrin in inducing postsynaptic specializations at neuromuscular junctions has been well characterized. Increasing evidence suggests that agrin is also involved in neuronal development. In this study,we found that agrin inhibited neurite extension and, more importantly, a gradient of agrin induced repulsive growth-cone turning in cultured Xenopus spinal neurons. Incubation with a neutralizing antibody to agrin or expression of the extracellular domain of muscle-specific kinase, a component of the agrin receptor complex, abolished these effects of agrin. Agrin-induced repulsive growth-cone turning requires the activity of PI3-kinase and Ca2+ signaling. In addition, the expression of dominant-negative Rac1 inhibited neurite extension and blocked agrin-mediated growth-cone turning. Taken together, our findings suggest that agrin regulates neurite extension and provide evidence for an unanticipated role of agrin in growth-cone steering in developing neurons.
BDNF stabilizes synapses and maintains the structural complexity of optic axons in vivo
Authors: Hu Bing , Nikolakopoulou Angeliki Maria , Cohen-Cory Susana
Published: September 2005

Brain-derived neurotrophic factor (BDNF) modulates synaptic connectivity by increasing synapse number and by promoting activity-dependent axon arbor growth. Patterned neuronal activity is also thought to influence the morphological maturation of axonal arbors by directly influencing the stability of developing synapses. Here, we used in vivo time-lapse imaging to examine the relationship between synapse stabilization and axon branch stabilization, and to better understand the participation of BDNF in synaptogenesis. Green fluorescent protein (GFP)-tagged synaptobrevin II was used to visualize presynaptic specializations in individual DsRed2-labeled Xenopus retinal axons arborizing in the optic tectum. Neutralizing endogenous tectal BDNF with function-blocking antibodies significantly enhanced GFP-synaptobrevin cluster elimination, a response that was paralleled by enhanced branch elimination. Thus, synapse dismantling was associated with axon branch pruning when endogenous BDNF levels were reduced. To obtain a second measure of the role of BDNF during synapse stabilization, we injected recombinant BDNF in tadpoles with altered glutamate receptor transmission in the optic tectum. Tectal injection of the NMDA receptor antagonists APV or MK801 transiently induced GFP-synaptobrevin cluster dismantling, but did not significantly influence axon branch addition or elimination. BDNF treatment rescued synapses affected by NMDA receptor blockade: BDNF maintained GFP-synaptobrevin cluster density by maintaining their addition rate and rapidly inducing their stabilization. Consequently, BDNF influences synaptic connectivity in multiple ways, promoting not only the morphological maturation of axonal arbors, but also their stabilization, by a mechanism that influences both synapses and axon branches.
Timed mutation and cell-fate mapping reveal reiterated roles ofTbx1during embryogenesis, and a crucial function during segmentation of the pharyngeal system via regulation of endoderm expansion
Authors: Xu Huansheng , Cerrato Fabiana , Baldini Antonio
Published: September 2005

The definition of time-specific requirements for a developmental gene can pinpoint the processes within which the gene is involved and can reveal potential late functions in structures and organs that fail to develop in germline mutants. Here, we show the first systematic time-course deletion, in parallel with timed cell fate mapping, of a developmentally crucial gene, Tbx1, during mouse embryogenesis. Tbx1 mouse mutants model DiGeorge syndrome, a disorder of pharyngeal and cardiovascular development. Results revealed different time requirements for the development of individual structures, as well as multiple and time-distinct roles during the development of the same organ or system. We also show that Tbx1 is required throughout pharyngeal segmentation for the regulation of endoderm expansion,thus this is the first gene implicated directly in this process. A genetic-based blueprint of crucial developmental times for organs and systems should be a valuable asset for our understanding of birth defect pathogenesis.
Ihh controls cartilage development by antagonizing Gli3, but requires additional effectors to regulate osteoblast and vascular development
Authors: Hilton Matthew J. , Tu Xiaolin , Cook Julie , Hu Hongliang , Long Fanxin
Published: September 2005

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development, including proliferation and maturation of chondrocytes,osteoblast development and cartilage vascularization. Although it is known that Gli transcription factors are key effectors of hedgehog signaling, it has not been established which Gli protein mediates Ihh activity in skeletal development. Here, we show that removal of Gli3 in Ihh-null mouse embryos restored normal proliferation and maturation of chondrocytes,but only partially rescued the defects in osteoblast development and cartilage vascularization. Remarkably, in both Ihh-/- and Ihh-/-; Gli3-/- embryos,vascularization promoted osteoblast development in perichondrial progenitor cells. Our results not only establish Gli3 as a critical effector for Ihh activity in the developing skeleton, but also identify an osteogenic role for a vasculature-derived signal, which integrates with Ihh and Wnt signals to determine the osteoblast versus chondrocyte fate in the mesenchymal progenitors.
HB-EGF promotes epithelial cell migration in eyelid development
Authors: Mine Naoki , Iwamoto Ryo , Mekada Eisuke
Published: September 2005

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR)and ERBB4. Here, we show that HB-EGF-EGFR signaling is involved in eyelid development. HB-EGF expression is restricted to the tip of the leading edge of the migrating epithelium during eyelid closure in late gestation mouse embryos. Both HB-EGF null (HBdel/del) and secretion-deficient(HBuc/uc) mutant embryos exhibited delayed eyelid closure, owing to slower leading edge extension and reduced actin bundle formation in migrating epithelial cells. No changes in cell proliferation were observed in these embryos. In addition, activation of EGFR and ERK was decreased in HBdel/del eyelids. Crosses between HBdel/del mice and waved 2 mice, a hypomorphic EGFR mutant strain, indicate that HB-EGF and EGFR interact genetically in eyelid closure. Together with our data showing that embryos treated with an EGFR-specific kinase inhibitor phenocopy HBdel/del embryos, these data indicate that EGFR mediates HB-EGF-dependent eyelid closure. Finally, analysis of eyelid closure in TGFα-null mice and in HB-EGF and TGFα double null mice revealed that HB-EGF and TGFα contribute equally to and function synergistically in this process. These results indicate that soluble HB-EGF secreted from the tip of the leading edge activates the EGFR and ERK pathway, and that synergy with TGFα is required for leading edge extension in epithelial sheet migration during eyelid closure.
Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis
Authors: Gachet Yannick , Hyams Jeremy S.
Published: September 2005

In the fission yeast, Schizosaccharomyces pombe, uptake of the fluorescent styryl dye FM4-64 via the endocytic pathway to the vacuole was localised to the poles of growing, interphase cells and to the cell equator during cell division, regions of cell wall deposition that are rich in actin. When the pattern of growth or the plane of cytokinesis was altered, the relationship between the actin cytoskeleton and the site of endocytosis was maintained. Transfer of the label to the vacuolar membrane was dependent upon the Rab GTPase Ypt7 and, hence, vesicle fusion. Endocytic vesicles transiently colocalised with actin patches and endocytosis was inhibited in mutants that affected actin patch integrity and by the actin inhibitor latrunculin A. Concentrations of latrunculin that removed actin cables but left patches unaffected had no effect on endocytosis at the poles, but abolished endocytosis at the cell equator. Equatorial, but not polar, endocytosis was also inhibited in cells lacking the formin For3 (which have selectively destabilised actin cables), in mutants of the exocyst complex and in cells treated with brefeldin A. Differential effects on endocytosis at the cell poles and equator were also observed in the actin mutant cps8 and the Arp2/3 complex mutant arp2. The redirection of endocytosis from the cell poles to the cell equator in M phase coincided with the anaphase separation of sister chromatids and was abolished in the septation initiation network (SIN) mutants cdc7, sid1 and sid2, demonstrating that the spatial reorganisation of the endocytic pathway in the S. pombe cell cycle requires a functional SIN pathway. We conclude that endocytosis in fission yeast has two distinct components, both of which are actin-based, but which are mechanistically distinct, as well as being spatially and temporally separated in the S. pombe cell cycle.
Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress
Authors: May Noah R. , Thomer Marguerite , Murnen Katherine F. , Calvi Brian R.
Published: September 2005

The regulation of a pre-replicative complex (pre-RC) at origins ensures that the genome is replicated only once per cell cycle. Cdt1 is an essential component of the pre-RC that is rapidly degraded at G1-S and also inhibited by Geminin (Gem) protein to prevent re-replication. We have previously shown that destruction of the Drosophila homolog of Cdt1, Double-parked (Dup), at G1-S is dependent upon cyclin-E/CDK2 and important to prevent re-replication and cell death. Dup is phosphorylated by cyclin-E/Cdk2, but this direct phosphorylation was not sufficient to explain the rapid destruction of Dup at G1-S. Here, we present evidence that it is DNA replication itself that triggers rapid Dup destruction. We find that a range of defects in DNA replication stabilize Dup protein and that this stabilization is not dependent on ATM/ATR checkpoint kinases. This response to replication stress was cell-type specific, with neuroblast stem cells of the larval brain having the largest increase in Dup protein. Defects at different steps in replication also increased Dup protein during an S-phase-like amplification cell cycle in the ovary, suggesting that Dup stabilization is sensitive to DNA replication and not an indirect consequence of a cell-cycle arrest. Finally, we find that cells with high levels of Dup also have elevated levels of Gem protein. We propose that, in cycling cells, Dup destruction is coupled to DNA replication and that increased levels of Gem balance elevated Dup levels to prevent pre-RC reformation when Dup degradation fails.
Implication of Pkc1p protein kinase C in sustaining Cln2p level and polarized bud growth in response to calcium signaling inSaccharomyces cerevisiae
Authors: Mizunuma Masaki , Hirata Dai , Miyakawa Tokichi
Published: September 2005

Protein kinase C, a highly conserved signaling molecule among eukaryotes, has been implicated in the regulation of cellular processes such as cell proliferation and polarized growth. In Saccharomyces cerevisiae, the unique protein kinase C Pkc1p is thought to have multiple functions, including the activation of the Mpk1p (Slt2p) MAP kinase pathway, which is essential for cell wall construction and bud emergence. However, little is known about the other functions of Pkc1p. In the course of screening for the mutants that suppress the Ca2+-sensitivity phenotype of the Ca2+-sensitive strain zdsΔ, we isolated a novel mutant allele (scz6/pkc1-834) of PKC1. Unlike the previously characterized PKC1 allele stt1-1, heat-shock-induced Mpk1p activation and cell-wall integrity were not impaired in the pkc1-834 mutant. By contrast, the mutant was defective in the maintenance of Ca2+-induced F-actin polarization in a manner independent of Mpk1p activation. This phenotype was caused by a decreased expression level of the G1 cyclin Cln2p. The Rho1 small G protein molecular switch was suggested to be involved in the novel Pkc1p function. The Pkc1p novel function was required for posttranscriptional upregulation of Cln2p and appeared to be important for the coordinated regulation of polar bud growth and the cell cycle.
pH-specific sequestration of phosphoglucose isomerase/autocrine motility factor by fibronectin and heparan sulphate
Authors: Lagana Annick , Goetz Jacky G. , Y Nathalie , Altschuler Yoram , Nabi Ivan R.
Published: September 2005

Phosphoglucose isomerase (PGI) is a glycolytic enzyme that moonlights as a cytokine under the aliases autocrine motility factor (AMF), neuroleukin and maturation factor. The cytokine function of PGI/AMF targets multiple cell types however mechanisms that regulate and sequester this ubiquitous, circulating cytokine remain largely unidentified. PGI/AMF is shown here to exhibit fibronectin (FN)-dependent cell surface association at both neutral and acid pH. Direct PGI/AMF binding to FN and fluorescence resonance energy transfer (FRET) between PGI/AMF and FN were detected only at pH 5. At neutral pH, the interaction of PGI/AMF with FN is receptor-mediated requiring prior clathrin-dependent endocytosis. PGI/AMF and FN do not co-internalize and PGI/AMF undergoes a second round of endocytosis upon recycling to the plasma membrane indicating that recycling PGI/AMF receptor complexes associate with FN fibrils. Heparan sulphate does not affect cell association of PGI/AMF at neutral pH but enhances the FN-independent cell surface association of PGI/AMF at acid pH identifying two distinct mechanisms for PGI/AMF sequestration under acidic conditions. However, only PGI/AMF sequestration by FN at acid pH was able to stimulate cell motility upon pH neutralization identifying FN as a pH-dependent cytokine trap for PGI/AMF. The multiple ways of cellular association of PGI/AMF may represent acquired mechanisms to regulate and harness the cytokine function of PGI/AMF.
Sulphonylurea receptors differently modulate ICC pacemaker Ca2+ activity and smooth muscle contractility
Authors: Nakayama Shinsuke , Ohya Susumu , Liu Hong-Nian , Watanabe Toshiya , Furuzono Shinji , Wang Jing , Nishizawa Yuji , Aoyama Masahiro , Murase Naruhiko , Matsubara Tatsuaki , Ito Yasushi , Imaizumi Yuji , Kajioka Shunichi
Published: September 2005

Appropriate gastrointestinal motility is essential to properly control the body energy level. Intracellular Ca2+ ([Ca2+]i) oscillations in interstitial cells of Cajal (ICCs; identified with c-Kit immunoreactivity) are considered to be the primary mechanism for the pacemaker activity in gastrointestinal motility. In the present study, RT-PCR examinations revealed predominant expression of the type 1 isoform of sulphonylurea receptors (SUR1) in ICCs of the mouse ileum, but expression of SUR2 was predominant in smooth muscle. In cell clusters prepared from the same tissue, smooth muscle contractility and pacemaker [Ca2+]i activity in ICCs were found to be differentially modulated by KATP channel openers and sulphonylurea compounds, in accordance with the expression of SUR isoforms. 1 μM cromakalim nearly fully suppressed the mechanical activity in smooth muscle, whereas ICC pacemaker [Ca2+]i oscillations persisted. Greater concentrations (∼10 μM) of cromakalim attenuated pacemaker [Ca2+]i oscillations. This effect was not reversed by changing the reversal potential of K+, but was prevented by glibenclamide. Diazoxide at 30 μM terminated ICC pacemaker [Ca2+]i oscillations, but again treatment with high extracellular K+ did not restore them. These results suggest that SUR can modulate pacemaker [Ca2+]i oscillations via voltage-independent mechanism(s), and also that intestinal pacemaking and glucose control are closely associated with SUR.
Accumulation of Werner protein at DNA double-strand breaks in human cells
Authors: Lan Li , Nakajima Satoshi , Komatsu Kenshi , Nussenzweig Andre , Shimamoto Akira , Oshima Junko , Yasui Akira
Published: September 2005

Werner syndrome is an autosomal recessive accelerated-aging disorder caused by a defect in the WRN gene, which encodes a member of the RecQ family of DNA helicases with an exonuclease activity. In vitro experiments have suggested that WRN functions in several DNA repair processes, but the actual functions of WRN in living cells remain unknown. Here, we analyzed the kinetics of the intranuclear mobilization of WRN protein in response to a variety of types of DNA damage produced locally in the nucleus of human cells. A striking accumulation of WRN was observed at laser-induced double-strand breaks, but not at single-strand breaks or oxidative base damage. The accumulation of WRN at double-strand breaks was rapid, persisted for many hours, and occurred in the absence of several known interacting proteins including polymerase β, poly(ADP-ribose) polymerase 1 (PARP1), Ku80, DNA-dependent protein kinase (DNA-PKcs), NBS1 and histone H2AX. Abolition of helicase activity or deletion of the exonuclease domain had no effect on accumulation, whereas the presence of the HRDC (helicase and RNaseD C-terminal) domain was necessary and sufficient for the accumulation. Our data suggest that WRN functions mainly at DNA double-strand breaks and structures resembling double-strand breaks in living cells, and that an autonomous accumulation through the HRDC domain is the initial response of WRN to the double-strand breaks.
RNA Polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product
Authors: Corbi Nicoletta , Bruno Tiziana , De Angelis Roberta , Di Padova Monica , Libri Valentina , Di Certo Maria Grazia , Spinardi Laura , Floridi Aristide , Fanciulli Maurizio , Passananti Claudio
Published: September 2005

Here, we show that the subcellular localization of α-like RNA polymerase II core subunit 3 (RPB3) is regulated during muscle differentiation. We have recently demonstrated that the expression of RPB3 is regulated during muscle differentiation and that, inside RNA polymerase II (RNAP II), it is directly involved in contacting regulatory proteins such as the myogenic transcription factor Myogenin and activating transcription factor ATF4. We show for the first time, that RPB3, in addition to its presence and role inside the RNAP II core enzyme, accumulates in the cytoplasm of cycling myogenic cells and migrates to the nucleus upon induction of the differentiation program. Furthermore, using human RPB3 as bait in a yeast two-hybrid system, we have isolated a novel RPB3 cytoplasmic interacting protein, HCR. HCR, previously identified as α-helix coiled-coil rod homologue, is one of the psoriasis vulgaris (PV) candidate genes. In cycling myogenic C2C7 cells, we show that the RPB3 protein directly interacts with HCR within the cytoplasm. Finally, knocking down HCR expression by RNA interference, we demonstrate that HCR acts as cytoplasmic docking site for RPB3.
Antiandrogens prevent stable DNA-binding of the androgen receptor
Authors: Farla Pascal , Hersmus Remko , Trapman Jan , Houtsmuller Adriaan B.
Published: September 2005

The androgen receptor (AR) is essential for development of the male gender and in the growth of the majority of prostate cancers. Agonists as well as most antagonists induce translocation of the receptor to the nucleus, whereas only agonists can activate AR function. Antagonists are therefore used in the therapy of metastasized prostate cancer. To obtain insight into the mechanism by which antagonists block AR function in living cells, we studied nuclear mobility and localization of green fluorescent protein (GFP)-tagged AR in the presence of either the agonist R1881 or the antagonists bicalutamide and hydroxyflutamide. As controls we investigated a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with broadened ligand specificity. We demonstrate that in the presence of R1881, AR localizes in numerous intranuclear foci and, using complementary fluorescence recovery after photobleaching (FRAP) approaches and computer modelling, that a fraction of AR (∼10-15%) is transiently immobilized in a DNA-binding-dependent manner (individual ARs being immobile for ∼45 seconds). By contrast, antagonist-bound GFP-AR showed no detectable immobile fraction and the mobility was similar to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating that antagonists do not induce the relatively stable DNA-binding-dependent immobilization observed with agonist-bound AR. Moreover, in the presence of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed in the nucleus. Binding of bicalutamide and hydroxyflutamide to GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar mobility and heterogeneous nuclear distribution as observed for R1881-liganded GFP-AR. The live cell studies indicate that the investigated antagonists interfere with events early in the transactivation function of the AR.
Selective nucleotide-release from dense-core granules in insulin-secreting cells
Authors: Obermüller Stefanie , Lindqvist Anders , Karanauskaite Jovita , Galvanovskis Juris , Rorsman Patrik , Barg Sebastian
Published: September 2005

Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by ∼0.1 and ∼2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.
The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability
Authors: Petkovic Maja , Dietschy Tobias , Freire Raimundo , Jiao Renjie , Stagljar Igor
Published: September 2005

Rothmund-Thomson syndrome (RTS) is a human genetic disorder characterized by genome instability, cancer susceptibility and premature aging. The gene defective in a subset of RTS cases, RECQL4, encodes a member of the RecQ family of DNA helicases. To better define the function of the RECQL4 protein, we have determined its subcellular localization. We have raised antibodies against the N- and C-terminal parts of RECQL4 and could show that in various human cells endogenous RECQL4 forms discrete nuclear foci that colocalize with promyelotic leukaemia protein (PML). The number of foci and their colocalization with PML does not significantly change after induction of different types of DNA damages. Silencing of RECQL4 expression by siRNA causes a significant reduction in RECQL4 nuclear foci formation. Furthermore, we demonstrate that RECQL4 foci coincide with foci formed by human Rad51 and regions of single-stranded DNA after induction of DNA double-strand breaks. In agreement with this, we also show that RECQL4 and Rad51 form a complex in human cells. Our findings suggest a role for RECQL4 in the repair of DNA double-strand breaks by homologous recombination and shed new light onto RECQL4's function in human cells.
Viral oncoprotein-induced mislocalization of select PDZ proteins disrupts tight junctions and causes polarity defects in epithelial cells
Authors: Latorre Isabel J. , Roh Michael H. , Frese Kristopher K. , Weiss Robert S. , Margolis Ben , Javier Ronald T.
Published: September 2005

The development of human cancers is frequently associated with a failure of epithelial cells to form tight junctions and to establish proper apicobasal polarity. Interestingly, the oncogenic potential of the adenovirus E4-ORF1 protein correlates with its binding to the cellular PDZ proteins MUPP1, MAGI-1, ZO-2 and SAP97, the first three of which assemble protein complexes at tight junctions. Given that E4-ORF1 sequesters these three PDZ proteins in the cytoplasm of fibroblasts, we postulated that E4-ORF1 would inhibit tight junction formation in epithelial cells. Providing further support for this idea, we identified MUPP1-related PATJ, a key component of the tight junction-associated CRB3-PALS1-PATJ polarity complex, as a new PDZ-protein target for both the E4-ORF1 and high-risk human papillomavirus type 18 E6 oncoproteins. Moreover, in epithelial cells, E4-ORF1 blocked the tight junction localization of PATJ and ZO-2, as well as their interacting partners, and disrupted both the tight junction barrier and apicobasal polarity. These significant findings expose a direct link between the tumorigenic potential of E4-ORF1 and inactivation of cellular PDZ proteins involved in tight junction assembly and polarity establishment.
Disassembling Single-walled Carbon Nanotube Bundles by Dipole/Dipole Electrostatic Interactions
Authors: Fugetsu Bunshi , Han Wenhai , Endo Noriaki , Kamiya Yuichi , Okuhara Toshio
Published: September 2005

Dipole/dipole electrostatic attraction was used for the first time for disassembling the single-walled carbon nanotube (SWCNT) bundles. A self-assembled zwitterionic monolayer (SAZM) was created by modification of the SWCNT bundles with a sulfonate/quaternary-ammonium type of zwitterionic surfactants. The headgroups of the SAZM align at the terminal-end and form anti-parallel doublets. Dipole/dipole electrostatic attractions occurred readily as the SAZM approached to another. SWCNT bundles have been disassembled into individual tubes because of the strong dipole/dipole attractions. Large quantities of unbundled single-walled carbon nanotubes (SWCNTs) were obtained within a single step by this method. Concentric circular membranes containing the unbundled SWCNTs were prepared. The unbundled SWCNTs were found to have tendencies to curve around the circular direction.
Capillary Forces between Planar Anchoring Surfaces in the Isotropic Phase of a Nematic Liquid Crystal
Authors: Shinto Hiroyuki , Kobayashi Kiwamu , Hyodo Terumasa , Ishida Naoyuki , Higashitani Ko
Published: September 2005

The interaction forces between the planar anchoring surfaces of a carbon microsphere and a graphite plate immersed in 8CB liquid crystal have been measured at different temperatures above the nematic–isotropic transition point, using an atomic force microscopy. The attractive force was observed between the surfaces when they came closer. The magnitude and the range of the attractive force increased with decreasing temperature. This attractive force is attributable to the confinement-induced phase separation of 8CB.
A New One-pot Synthesis of Thiocarbamates from Isocyanates and Disulfides in the Presence of Zn/AlCl3 System
Authors: Movassagh Barahman , Zakinezhad Yousef
Published: September 2005

A novel method has been developed for the synthesis of S-alkyl(aryl) thiocarbamates. The route involves, first, the formation of zinc thiolates by reductive cleavage of disulfides in the presence of Zn/AlCl3 system; then subsequent reaction of the thiolate anions with isocyanates gives the desired thiocarbamates in high to excellent yields.
41Ca and Accelerator Mass Spectrometry to Monitor Calcium Metabolism in End Stage Renal Disease Patients
Authors: Fitzgerald Robert L , Hillegonds Darren J , Burton Douglas W , Griffin Terrance L , Mullaney Scott , Vogel John S , Deftos Leonard J , Herold David A
Published: September 2005

Background: Monitoring bone resorption with measurements of bone density and biochemical markers is indirect. We hypothesized that bone resorption can be studied directly by serial measurements of the ratio 41Ca/Ca in serum after in vivo labeling of calcium pools with 41Ca. We report the preparation of an intravenous 41Ca dose suitable for humans, an analytical method for determining 41Ca/Ca isotope ratios in biological samples, and studies in human volunteers.

Methods: 41Ca was formulated and aliquoted into individual vials, and to the extent possible, the 41Ca doses were tested according to US Pharmacopeia (USP) guidelines. A 10 nCi dose of 41Ca was administered intravenously to 4 end stage renal disease (ESRD) patients on hemodialysis and 4 healthy control individuals. Distribution kinetics were determined over 168 days. Calcium was isolated with 3 precipitation steps and a cation-exchange column, and 41Ca/Ca ratios in serum were then measured by accelerator mass spectrometry.

Results: The dosing solution was chemically and radiologically pure, contained <0.1 endotoxin unit/mL, and passed USP sterility tests. Quantification of 41Ca/Ca ratios was linear from 6 × 10−14 to 9.1 × 10−10. The run-to-run imprecision (as CV) of the method was 4% at 4.6 × 10−11 and 6% at 9.1 × 10−10. The area under the curve of 41Ca in the central compartment vs time was significantly less for ESRD patients than for controls (P <0.005).

Conclusions: Isotope ratios spanning 5 orders of magnitude can be measured by accelerator mass spectrometry with excellent precision in the range observed in samples collected from patients who have received 10 nCi of 41Ca. The 41Ca at this dose caused no adverse effects in 8 volunteers. This is the first report of the use of 41Ca to monitor differences in bone turnover between healthy individuals and ESRD patients.
Automated Spectrophotometric Analysis of Mitochondrial Respiratory Chain Complex Enzyme Activities in Cultured Skin Fibroblasts
Authors: Kramer Karen A , Oglesbee Devin , Hartman Stacy J , Huey Joe , Anderson Bambi , Magera Mark J , Matern Dietrich , Rinaldo Piero , Robinson Brian H , Cameron Jessie M , Hahn Si Houn
Published: September 2005

Background: Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial.

Method: We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities.

Results: A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RCC was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%–18%.

Conclusions: With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.
PARK7 and Nucleoside Diphosphate Kinase A as Plasma Markers for the Early Diagnosis of Stroke
Authors: Allard Laure , Burkhard Pierre R , Lescuyer Pierre , Burgess Jennifer A , Walter Nadia , Hochstrasser Denis F , Sanchez Jean-Charles
Published: September 2005

Background: Plasma markers for stroke could be useful in diagnosis and prognosis and in prediction of response of stroke patients to therapy. PARK7 and nucleoside diphosphate kinase A (NDKA) are increased in human postmortem cerebrospinal fluid (CSF), a model of global brain insult, suggesting that measurement in CSF and, more importantly, in plasma may be useful as a biomarker of stroke.

Methods: We used ELISA to measure PARK7 and NDKA in plasma in 3 independent European and North American retrospective studies encompassing a total of 622 stroke patients and 165 control individuals.

Results: Increases in both biomarkers were highly significant, with sensitivities of 54%–91% for PARK7 and 70%–90% for NDKA and specificities of 80%–97% for PARK7 and 90%–97% for NDKA. The concentrations of both biomarkers increased within 3 h of stroke onset.

Conclusions: PARK7 and NDKA may be useful plasma biomarkers for the early diagnosis of stroke. In addition, this study demonstrated the utility of analysis of postmortem CSF proteins as a first step in the discovery of plasma markers of ischemic brain injury.