bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Murine Model of Autoimmune Thyroiditis Induced with Homologous Mouse Thyroid Peroxidase
Authors: Ng H. P. , Paul Banga J. , Kung Annie W. C.
Published: November 2003

Autoimmune thyroid disease (AITD) is a common autoimmune disease. Thyroid peroxidase (TPO) is a well characterized autoantigen in AITD. Autoantibodies and autoreactive T lymphocytes to TPO are believed to play a major role in the pathogenesis of lymphocytic thyroiditis. To understand the pathogenic mechanisms of AITD and the role of TPO, we have established a mouse model of lymphocytic thyroiditis by immunizing C57Bl/6 (H-2b), CBA (H-2k), and C57Bl/6 × CBA F1 mice with recombinant murine TPO (rmTPO) ectodomain comprising amino acid residue 1–837 produced in Escherichia coli. Mice were immunized with 30 μg purified ectodomain in complete Freund’s adjuvant. Antibodies against rmTPO were detected in the serum of all mice from day 21 onward. Draining lymph node cells from rmTPO-immunized animals showed dose-dependent proliferation to TPO stimulation. Mice killed at d 50 and 90 revealed variable degrees of thyroiditis with infiltration of mononuclear cells and destruction of thyroid follicles. C57Bl/6 and the F1 mice, in comparison with CBA mice, showed a greater degree of thyroiditis. There was a lack of correction between the intensity of thyroiditis and the anti-TPO response. Immunotyping of the thyroid cellular infiltrates showed predominantly CD4+ T cells and B220+ B cells but scanty CD8+ T cells. None of the control mice injected with the purified fusion partner developed anti-TPO antibodies and thyroiditis. In conclusion, a genuine autoimmune mouse model of lymphocytic thyroiditis was established using autologous mouse TPO. This new model induced with autologous TPO will lead to a better understanding of the mechanisms in destructive thyroiditis and will assist in the development of new strategies for modulating the pathogenic immune response.
Insulin-Like Growth Factor I Increases Rat Peptide YY Promoter Activity through Sp1 Binding Sites
Authors: Wang Guiyun , Leiter Andrew B. , Englander Ella W. , Greeley George H.
Published: November 2003

Studies in rodents demonstrate that the mitogen, IGF-I, stimulates intestinal peptide YY (PYY) expression. To investigate whether the stimulatory influence of IGF-I is exerted at the level of gene transcription, rat PYY 5′-upstream sequences (−2800/+37 bp, −770/+37 bp, −127/+37 bp) fused to the firefly luciferase (luc) reporter gene were transfected into rat pheochromocytoma cells (PC12) and luc activity measured after IGF-I treatment. IGF-I increased transcriptional activity of all constructs similarly; the PYY (−127/+37 bp)-luc construct was used in subsequent experiments. IGF-I increased PYY (−127/+37 bp)-luc activity in a time- and dose-dependent fashion. Sequence analysis detected five putative Sp1 binding sites in the −127/+37-bp sequence. EMSA and supershift experiments using two oligonucleotide fragments of the −127/+37 region showed that Sp1 and Sp3 proteins bound to putative Sp1 sites. Overexpression of Sp1 greatly increased PYY (−127/+37 bp)-luc activity and site-directed mutagenesis of putative Sp1 binding sites decreased basal and IGF-I-induced elevations in PYY (−127/+37 bp)-luc activity. IGF-I treatment also increased Sp1 protein levels and binding activity. Blockade of the IGF-I receptor (IGF-IR) with an IGF-IR antibody decreased the stimulatory influence of IGF-I on Sp1 protein levels and PYY (−127/+37 bp)-luc activity. Together, these findings indicate that IGF-I functions as a positive regulator of PYY gene expression and that the stimulatory effect may be mediated by Sp1 proteins that bind to the proximal PYY promoter region.
Androgen Contributes to Gender-Related Cardiac Hypertrophy and Fibrosis in Mice Lacking the Gene Encoding Guanylyl Cyclase-A
Authors: Li Yuhao , Kishimoto Ichiro , Saito Yoshihiko , Harada Masaki , Kuwahara Koichiro , Izumi Takehiko , Hamanaka Ichiro , Takahashi Nobuki , Kawakami Rika , Tanimoto Keiji , Nakagawa Yasuaki , Nakanishi Michio , Adachi Yuichiro , Garbers David L. , Fukamizu Akiyoshi , Nakao Kazuwa
Published: November 2003

Myocardial hypertrophy and extended cardiac fibrosis are independent risk factors for congestive heart failure and sudden cardiac death. Before age 50, men are at greater risk for cardiovascular disease than age-matched women. In the current studies, we found that cardiac hypertrophy and fibrosis were significantly more pronounced in males compared with females of guanylyl cyclase-A knockout (GC-A KO) mice at 16 wk of age. These gender-related differences were not seen in wild-type mice. In the further studies, either castration (at 10 wk of age) or flutamide, an androgen receptor antagonist, markedly attenuated cardiac hypertrophy and fibrosis in male GC-A KO mice without blood pressure change. In contrast, ovariectomy (at 10 wk of age) had little effect. Also, chronic testosterone infusion increased cardiac mass and fibrosis in ovariectomized GC-A mice. None of the treatments affected cardiac mass or the extent of fibrosis in wild-type mice. Overexpression of mRNAs encoding atrial natriuretic peptide, brain natriuretic peptide, collagens I and III, TGF-β1, TGF-β3, angiotensinogen, and angiotensin converting enzyme in the ventricles of male GC-A KO mice was substantially decreased by castration. The gender differences were virtually abolished by targeted deletion of the angiotensin II type 1A receptor gene (AT1A). Neither castration nor testosterone administration induced any change in the cardiac phenotypes of double-KO mice for GC-A and AT1A. Thus, we suggest that androgens contribute to gender-related differences in cardiac hypertrophy and fibrosis by a mechanism involving AT1A receptors and GC-A.
Estrogen Regulation of Neurokinin B Gene Expression in the Mouse Arcuate Nucleus Is Mediated by Estrogen Receptor α
Authors: Dellovade Tammy L. , Merchenthaler Istvan
Published: November 2003

Neurokinin B (NKB) gene expression is elevated in the infundibular (arcuate) nucleus of the hypothalamus in postmenopausal women. Estrogen replacement decreases both the number of NKB mRNA-expressing neurons and the level of expression within individual cells. Similarly, NKB gene expression is elevated in ovariectomized rats and reduced after estrogen treatment. The actions of estrogen in the brain can be mediated via either estrogen receptor α (ERα) or estrogen receptor β (ERβ). In the rodent arcuate nucleus (ARC), more ERα- than ERβ-containing cells are present, suggesting that ERα might be directly responsible for estrogen regulation of NKB gene expression. However, an indirect effect via ERβ could not be ruled out. Here we used ERα knockout and ERβ knockout mice to identify the type of ER responsible for mediating estrogen action on NKB gene expression in the ARC. Using in situ hybridization histochemistry, we have found that estrogen treatment significantly reduced NKB gene expression in the ARC of ovariectomized ERβ knockout mice, but had no effect on NKB mRNA levels in ERα knockout mice. These data indicate that ERα mediates the increase in NKB gene expression associated with ovariectomy in rodents and might also be responsible for the increase in NKB in postmenopausal women.
Connective Tissue Growth Factor Is Up-Regulated in the Diabetic Retina: Amelioration by Angiotensin-Converting Enzyme Inhibition
Authors: Tikellis Christos , Cooper Mark E. , Twigg Stephen M. , Burns Wendy C. , Tolcos Mary
Published: November 2003

Connective tissue growth factor (CTGF) has been postulated to have prosclerotic and angiogenic properties. The aim of this present study was to characterize retinal CTGF expression in the absence and presence of diabetes and in the context of treatment with the angiotensin-converting enzyme (ACE) inhibitor, perindopril. Retinas were obtained from control, diabetic, and diabetic plus perindopril-treated (3 mg/d) rats. CTGF gene expression was quantitated by RT-PCR and localized by in situ hybridization. CTGF protein expression was analyzed by Western blotting and localized by immunohistochemistry. Diabetes was associated with a greater than 2-fold increase in CTGF mRNA levels, which was attenuated by perindopril treatment. CTGF immunoreactivity was increased almost 2-fold in diabetes and was ameliorated by the ACE inhibitor perindopril. By in situ hybridization and immunohistochemistry, the major site of CTGF gene expression in the retina of diabetic rats was the ganglion cell layer. Based on the known in vivo effects of CTGF, it is postulated that this growth factor plays a pivotal role in mediating diabetes-associated retinal pathology. Furthermore, the protective effects of ACE inhibitors on retinal pathology may partly be mediated via effects on retinal CTGF expression.
Glucose Sensitivity and Metabolism-Secretion Coupling Studied during Two-Year Continuous Culture in INS-1E Insulinoma Cells
Authors: Merglen Arnaud , Theander Sten , Rubi Blanca , Chaffard Gaelle , Wollheim Claes B. , Maechler Pierre
Published: November 2003

Rat insulinoma-derived INS-1 cells constitute a widely used β-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E β-cell phenotype over 116 passages (no. 27–142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40–100 with average insulin contents of 2.30 ± 0.11 μg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca2+, and mitochondrial Ca2+ levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A KATP current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native β-cells, tolbutamide induced electrical activity, indicating that the KATPconductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable β-cell model.
Adiponectin Ameliorates Dyslipidemia Induced by the Human Immunodeficiency Virus Protease Inhibitor Ritonavir in Mice
Authors: Xu Aimin , Yin Shinan , Wong LaiChing , Weng Chan Kok , Lam Karen S. L.
Published: November 2003

Although the clinical application of HIV protease inhibitors (PIs) has markedly reduced HIV-related morbidity and mortality, it is now recognized that PI-based therapy often causes serious metabolic disorders, including hyperlipidemia and premature atherosclerosis. The etiology of these adverse effects remains obscure. Here, we demonstrate that deficiency of the fat-derived hormone adiponectin might play a role. The steady-state mRNA levels of the adiponectin gene and secretion of this protein from 3T3-L1 adipocytes are significantly decreased after treatment with several PIs (indinavir, nelfinavir, and ritonavir), with ritonavir having the greatest effect. Intragastric administration of ritonavir into mice decreases plasma concentrations of adiponectin and concurrently increases the plasma levels of triglyceride, free fatty acids, and cholesterol. Adiponectin replacement therapy markedly ameliorates ritonavir-induced elevations of triglyceride and free fatty acids. These beneficial effects of adiponectin are partly due to its ability to decrease ritonavir-induced synthesis of fatty acids and triglyceride, and to increase fatty acid combustion in the liver tissue. In contrast, adiponectin has little effect on ritonavir-induced hypercholesterolemia and hepatic cholesterol synthesis. These results suggest that hypoadiponectinemia is partly responsible for the metabolic disorders induced by HIV PIs, and adiponectin or its agonists might be useful for the treatment of these disorders.
Mechanisms of germ cell specification across the metazoans: epigenesis and preformation
Authors: Extavour Cassandra G. , Akam Michael
Published: November 2003

Germ cells play a unique role in gamete production, heredity and evolution. Therefore, to understand the mechanisms that specify germ cells is a central challenge in developmental and evolutionary biology. Data from model organisms show that germ cells can be specified either by maternally inherited determinants (preformation) or by inductive signals (epigenesis). Here we review existing data on 28 metazoan phyla, which indicate that although preformation is seen in most model organisms, it is actually the less prevalent mode of germ cell specification, and that epigenetic germ cell specification may be ancestral to the Metazoa.
Distinct sites in E-cadherin regulate different steps inDrosophilatracheal tube fusion
Authors: Lee Mihye , Lee Seungbok , Zadeh Alireza Dehghani , Kolodziej Peter A.
Published: November 2003

We have investigated how E-cadherin controls the elaboration of adherens junction associated cytoskeletal structures crucial for assembling tubular networks. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Shot, a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to β-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct,evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly.
Cct1, a phosphatidylcholine biosynthesis enzyme, is required forDrosophilaoogenesis and ovarian morphogenesis
Authors: Gupta Tripti , Schüpbach Trudi
Published: November 2003

Patterning of the Drosophila egg requires cooperation between the germline cells and surrounding somatic follicle cells. In order to identify genes involved in follicle cell patterning, we analyzed enhancer trap lines expressed in specific subsets of follicle cells. Through this analysis, we have identified tandem Drosophila genes homologous to CTP:phosphocholine cytidylyltransferase (CCT), the second of three enzymes in the CDP-choline pathway, which is used to synthesize phosphatidylcholine. Drosophila Cct1 is expressed at high levels in three specific subsets of follicle cells, and this expression is regulated, at least in part, by the TGF-β and Egfr signaling pathways. Mutations in Cct1 result in a number of defects, including a loss of germline stem cell maintenance,mispositioning of the oocyte, and a shortened operculum, suggesting that Cct1 plays multiple roles during oogenesis. In addition, Cct1 mutants display a novel branched ovariole phenotype,demonstrating a requirement for this gene during ovarian morphogenesis. These data provide the first evidence for a specific role for CCT, and thus for phosphatidylcholine, in patterning during development.
Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation
Authors: Dahlqvist Camilla , Blokzijl Andries , Chapman Gavin , Falk Anna , Dannaeus Karin , Ibâñez Carlos F. , Lendahl Urban
Published: November 2003

The bone morphogenetic protein (BMP) and Notch signaling pathways are crucial for cellular differentiation. In many cases, the two pathways act similarly; for example, to inhibit myogenic differentiation. It is not known whether this inhibition is caused by distinct mechanisms or by an interplay between Notch and BMP signaling. Here we demonstrate that functional Notch signaling is required for BMP4-mediated block of differentiation of muscle stem cells, i.e. satellite cells and the myogenic cell line C2C12. Addition of BMP4 during induction of differentiation dramatically reduced the number of differentiated satellite and C2C12 cells. Differentiation was substantially restored in BMP4-treated cultures by blocking Notch signaling using either theγ-secretase inhibitor L-685,458 or by introduction of a dominant-negative version of the Notch signal mediator CSL. BMP4 addition to C2C12 cells increased transcription of two immediate Notch responsive genes, Hes1 and Hey1, an effect that was abrogated by L-685,458. A 3 kb Hey1-promoter reporter construct was synergistically activated by the Notch 1 intracellular domain (Notch 1 ICD) and BMP4. The BMP4 mediator SMAD1 mimicked BMP activation of the Hey1 promoter. A synthetic Notch-responsive promoter containing no SMAD1 binding sites responded to SMAD1, indicating that DNA-binding activity of SMAD1 is not required for activation. Accordingly, Notch 1 ICD and SMAD1 interacted in binding experiments in vitro. Thus, the data presented here provide evidence for a direct interaction between the Notch and BMP signaling pathways, and indicate that Notch has a crucial role in the execution of certain aspects of BMP-mediated differentiation control.
Replicated anterior zeugopod (raz): a polydactylous mouse mutant with lowered Shh signaling in the limb bud
Authors: Krebs Ottheinz , Schreiner Claire M. , Scott William J. , Bell Sheila M. , Robbins David J. , Goetz John A. , Alt Heidi , Hawes Norm , Wolf Eckhard , Favor Jack
Published: November 2003

A unique limb phenotype is described in a radiation-induced mutant mouse resulting from an inversion of a proximal segment of chromosome 5. The limb phenotype in the homozygous mutant presents with two anterior skeletal elements in the zeugopod but no posterior bone, hence the name replicated anterior zeugopod, raz. The zeugopod phenotype is accompanied by symmetrical central polydactyly of hand and foot. The chromosomal inversion includes the Shh gene and the regulatory locus, located ∼1 Mb away, within the Lmbr1 gene. In homozygous mutants, the expression of Shh mRNA and Shh protein is severely downregulated to about 20% of wild-type limb buds, but Shh expression appears normal throughout the remainder of the embryo. Correspondingly, Gli3 expression is upregulated and posteriorly expanded in the raz/raz limb bud. We propose that the double anterior zeugopod and symmetrical central polydactyly are due to an increased and uniform concentration of the Gli3 repressor form because of lowered Shh signaling.
A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, theDrosophilasex determination master switch
Authors: Horabin Jamila I. , Walthall Sabrina , Vied Cynthia , Moses Michelle
Published: November 2003

The sex determination master switch, Sex-lethal(Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci)the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched(Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely,overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci.
The control of growth
Authors: Nijhout H. Frederik
Published: November 2003

The growth of a cell or tissue involves complex interactions between genes,metabolism, nutrition and hormones. Until recently, separate lines of investigation have concentrated in isolated sections of each of the many independent levels of growth control; the interactions within and between the diverse pathways that affect growth and size at the cellular, tissue and organismal level were little understood. However, new insights into the control of growth are now emerging in the context of signalling, ageing,evolution, cancer and nutrition. In particular, it is becoming clear that the insulin signaling network is a key player that integrates not only metabolism and the response to nutrition, but also the regulation of cell death, ageing and longevity, as well as the regulation of growth and body size.
A targeted deletion of the C-terminal end of titin, including the titin kinase domain, impairs myofibrillogenesis
Authors: Miller Gaynor , Musa Hanny , Gautel Matthias , Peckham Michelle
Published: November 2003

Titin is the largest protein known, and is essential for organising muscle sarcomeres. It has many domains with a variety of functions, and stretches from the Z-line to the M-line in the muscle sarcomere. Close to the M-line, titin contains a kinase domain, which is known to phosphorylate the Z-line protein telethonin in developing muscle (Mayans, O., van der Ven, P. F., Wilm, M., Mues, A., Young, P., Furst, D. O., Wilmanns, M. and Gautel, M. (1998) Nature 395, 863-869). This phosphorylation is thought to be important for initiating or regulating myofibrillogenesis. We used a gene-targeting approach in cultured myoblasts to truncate the titin gene so that the kinase domain and other domains downstream of the kinase were not expressed. We recovered cells in which one allele was targeted. We found that these cells expressed both the full-length and a truncated titin that was approximately 0.2 MDa smaller than the corresponding band from wild-type cells. Myofibrillogenesis in these cells was impaired, in that the myotubes were shorter, and the organisation of the muscle sarcomeres, M- and Z-lines was poorer than in wild-type cells. There was also an overall reduction in levels of titin and skeletal myosin expression. These results suggest that the activity of the titin kinase domain and downstream sequence are important in organising myofibrils both at the M- and the Z-line early in myofibrillogenesis.
Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization
Authors: Payne Christopher , Rawe Vanesa , Ramalho-Santos João , Simerly Calvin , Schatten Gerald
Published: November 2003

Fertilization is complete once the parental genomes unite, and requires the migration of the egg nucleus to the sperm nucleus (female and male pronuclei, respectively) on microtubules within the inseminated egg. Neither the molecular mechanism of pronucleus binding to microtubules nor the role of motor proteins in regulating pronuclear motility has been fully characterized, and the failure of zygotic development in some patients suggests that they contribute to human infertility. Based on the minus-end direction of female pronuclear migration, we propose a role for cytoplasmic dynein and dynactin in associating with the pronuclear envelope and mediating genomic union. Our results show that dynein intermediate and heavy chains preferentially concentrate around the female pronucleus, whereas dynactin subunits p150Glued, p50 and p62 localize to the surfaces of both pronuclei. Transfection of antibodies against dynein and dynactin block female pronuclear migration in zygotes. Both parthenogenetic activation in oocytes and microtubule depolymerization in zygotes significantly reduce the localization of dynein to the female pronucleus but do not inhibit the pronuclear association of dynactin. When immunoprecipitated from zygotes, p150Glued associates with nuclear pore complex proteins, as well as the intermediate filament vimentin and dynein. Antibodies against nucleoporins and vimentin inhibit pronuclear apposition when transfected into zygotes. We conclude that preferentially localized dynein and perinuclear dynactin associate with the nuclear pore complex and vimentin and are required to mediate genomic union. These data suggest a model in which dynein accumulates and binds to the female pronucleus on sperm aster microtubules, where it interacts with dynactin, nucleoporins and vimentin.
Specific sorting of the a1 isoform of the V-H+ATPase a subunit to nerve terminals where it associates with both synaptic vesicles and the presynaptic plasma membrane
Authors: Morel Nicolas , Dedieu Jean-Claude , Philippe Jean-Marc
Published: November 2003

Vacuolar H+ATPase (V-ATPase) accumulates protons inside various intracellular organelles, generating the electrochemical proton gradient required for many vital cellular processes. V-ATPase is a complex enzyme with many subunits that are organized into two domains. The membrane domain that translocates protons contains a proteolipid oligomer of several c subunits and a 100 kDa a subunit. Several a-subunit isoforms have been described that are important for tissue specificity and targeting to different membrane compartments, and could also result in the generation of V-ATPases with different functional properties. In the present report, we have cloned the Torpedo marmorata a1 isoform. This isoform was found to be addressed specifically to nerve endings, whereas VATPases in the neuron cell bodies contain a different a-subunit isoform. In nerve terminals, the V-ATPase membrane domain is present not only in synaptic vesicles but also in the presynaptic plasma membrane, where its density could reach 200 molecules μm–2. This V-ATPase interacts with VAMP-2 and with the SNARE complexes involved in synaptic vesicle docking and exocytosis.
RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate
Authors: Chang Chih-Jui , Goulding Sarah , Earnshaw William C. , Carmena Mar
Published: November 2003

DNA topoisomerase II (Topo II) is a major component of mitotic chromosomes and an important drug target in cancer chemotherapy, however, its role in chromosome structure and dynamics remains controversial. We have used RNAi to deplete Topo II in Drosophila S2 cells in order to carry out a detailed functional analysis of the role of the protein during mitosis. We find that Topo II is not required for the assembly of a functional kinetochore or the targeting of chromosomal passenger proteins, nonetheless, it is essential for anaphase sister chromatid separation. In response to a long-running controversy, we show that Topo II does have some role in mitotic chromatin condensation. Chromosomes formed in its absence have a 2.5-fold decrease in the level of chromatin compaction, and are morphologically abnormal. However, it is clear that the overall programme of mitotic chromosome condensation can proceed without Topo II. Surprisingly, in metaphase cells depleted of Topo II, one or more chromosome arms frequently stretch out from the metaphase plate to the vicinity of the spindle pole. This is not kinetochore-based movement, as the centromere of the affected chromosome is located on the plate. This observation raises the possibility that further unexpected functions for Topo II may remain to be discovered.
Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation
Authors: Trieselmann Nadia , Armstrong Sheri , Rauw Jennifer , Wilde Andrew
Published: November 2003

Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin β, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.
Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis
Authors: Coelho Paula A. , Queiroz-Machado Joana , Sunkel Claudio E.
Published: November 2003

Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin we have depleted DmSMC4, a subunit of the complex, from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. Our results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution.
Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis
Authors: Galperin Emilia , Sorkin Alexander
Published: November 2003

Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.
The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics
Authors: Emery Gregory , Parton Robert G. , Rojo Manuel , Gruenberg Jean
Published: November 2003

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility – two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.
β-Dystrobrevin interacts directly with kinesin heavy chain in brain
Authors: Macioce P. , Gambara G. , Bernassola M. , Gaddini L. , Torreri P. , Macchia G. , Ramoni C. , Ceccarini M. , Petrucci T. C.
Published: November 2003

β-Dystrobrevin, a member of the dystrobrevin protein family, is a dystrophin-related and -associated protein restricted to non-muscle tissues and is highly expressed in kidney, liver and brain. Dystrobrevins are now thought to play an important role in intracellular signal transduction, in addition to providing a membrane scaffold in muscle, but the precise role of β-dystrobrevin has not yet been determined. To study β-dystrobrevin's function in brain, we used the yeast two-hybrid approach to look for interacting proteins. Four overlapping clones were identified that encoded Kif5A, a neuronal member of the Kif5 family of proteins that consists of the heavy chains of conventional kinesin. A direct interaction of β-dystrobrevin with Kif5A was confirmed by in vitro and in vivo association assays. Co-immunoprecipitation with a monoclonal kinesin heavy chain antibody precipitated both α- and β-dystrobrevin, indicating that this interaction is not restricted to the β-dystrobrevin isoform. The site for Kif5A binding to β-dystrobrevin was localized in a carboxyl-terminal region that seems to be important in heavy chain-mediated kinesin interactions and is highly homologous in all three Kif5 isoforms, Kif5A, Kif5B and Kif5C. Pull-down and immunofluorescence experiments also showed a direct interaction between β-dystrobrevin and Kif5B. Our findings suggest a novel function for dystrobrevin as a motor protein receptor that might play a major role in the transport of components of the dystrophin-associated protein complex to specific sites in the cell.
Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration
Authors: Tufvesson Ellen , Westergren-Thorsson Gunilla
Published: November 2003

Biglycan and decorin are small chondroitin/dermatan sulphate proteoglycans in the extracellular matrix of connective tissue that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins.

We show for the first time that biglycan and decorin induce morphological and cytoskeletal changes in fibroblasts, resulting in an increase in migration. Biglycan changed the cell shape of fibroblasts with formation of long protruding filamentous processes. This was also seen for decorin but to a lesser extent. Using fluorescence staining of F-actin fibres it was possible to show that these long filamentous processes were supported by long thick bundles of actin, together with an induced formation of stress fibres after stimulation with biglycan and decorin. Moreover, a reorganisation of α-smooth muscle actin was clearly seen in these cultures. Decorin also stimulated α-smooth muscle actin expression in the cells. Using cDNA Atlas Arrays we were also able to show that the mRNA level of a number of the intracellular regulators and effectors involved in cell migration were increased. For example, the focal adhesion proteins paxillin and zyxin, and some of the small Rho GTPases such as RhoA, Rac1 and Cdc42 were upregulated. After treatment with biglycan or decorin, additional results showed an increased activation of RhoA (1.8- and 1.5-fold, respectively) and Rac1 (1.8- and 1.5-fold, respectively) after 15 minutes. These factors are known to be involved in fibroblast migration, and as expected a 1.3- to 1.6-fold increase in migration could be observed after stimulation with biglycan or decorin. This induced migration was caused by the core protein, as treatment with glycosaminoglycan chains alone did not have any effect.

In summary, these data indicate that biglycan- and decorin-induced fibroblast cytoskeletal and signalling changes result in an increased cell migration, and demonstrate their potential role in the remodelling process.
T-cell integrins: more than just sticking points
Authors: Hogg Nancy , Laschinger Melanie , Giles Katherine , McDowall Alison
Published: November 2003

T cells use integrins in essentially all of their functions. They use integrins to migrate in and out of lymph nodes and, following infection, to migrate into other tissues. At the beginning of an immune response, integrins also participate in the immunological synapse formed between T cells and antigen-presenting cells. Because the ligands for integrins are widely expressed, integrin activity on T cells must be tightly controlled. Integrins become active following signalling through other membrane receptors, which cause both affinity alteration and an increase in integrin clustering. Lipid raft localization may increase integrin activity. Signalling pathways involving ADAP, Vav-1 and SKAP-55, as well as Rap1 and RAPL, cause clustering of leukocyte function-associated antigen-1 (LFA-1; integrin αLβ2). T-cell integrins can also signal, and the pathways dedicated to the migratory activity of T cells have been the most investigated so far. Active LFA-1 causes T-cell attachment and lamellipodial movement induced by myosin light chain kinase at the leading edge, whereas RhoA and ROCK cause T-cell detachment at the trailing edge. Another important signalling pathway acts through CasL/Crk, which might regulate the activity of the GTPases Rac and Rap1 that have important roles in T-cell migration.
Selective Recovery of Metal Ions Based on Selective Sinking of Zeolite Modified with Octadecyl and Diethylenetriamine Groups
Authors: Nishimura Go , Shiraishi Yasuhiro , Hirai Takayuki
Published: November 2003

Zeolite modified with octadecyl and diethylenetriamine groups (ZOD), which floats on surface of water, sinks into the water via the formation of metal–ligand complex, owing to the increase in hydrophilicity. The degree of the sinking depends on the metal ions coordinated, allowing selective recovery of ZOD particles loading high concentration of specific metal ion.
The First Observation of an Atomic Scale Noncontact AFM Image of MoO3(010)
Authors: Suzuki Shushi , Ohminami Yusuke , Tsutsumi Tetsuya , Shoaib M M , Ichikawa Masaru , Asakura Kiyotaka
Published: November 2003

We have observed MoO3(010) surface on an atomic level by NC-AFM(Noncontact atomic force microscopy) for the first time. White arrays with 0.40 and 0.37 nm dimensions along [100] and [001], respectively, were observed, which corresponded to the topmost oxygen atoms of MoO3(010).
Hydrogen Bond between Water and the Phenyl Ring in the Structure of a Dipeptide H–Phe–Leu–NH2 at 90 K and the Structure-based Energy Estimations
Authors: Doi Mitsunobu , Asano Akiko , Yamamoto Daisuke
Published: November 2003

A water molecule forms a hydrogen bond with the aromatic π-electron system within the crystal structure of H–Phe–Leu–NH2 hydrochloride monosolvate. This water molecule located above the phenyl ring plane, 3.119 Å from the centroid of the ring. Ab initio energy calculations revealed the stabilization energy of the phenyl ring–water hydrogen bond to be 3.26 kcal/mol.
Endo–Endo Mode Intramolecular Reductive Cyclization of Cyclic 1,2-Bis(silylethynyl)benzenes
Authors: Yamaguchi Shigehiro , Miyasato Masataka , Tamao Kohei
Published: November 2003

Upon treatment of cyclic 1,2-bis(silylethynyl)benzenes with lithium naphthalenide, the intramolecular reductive cyclization proceeds in an endo–endo mode to produce 1,4-dilithio-2,3-disilylnaphthalenes, which are transformed into a series of 1,4-difunctionalized 2,3-disilylnaphthalenes.
Structures of a Single-component Palladium Complex with Extended TTF-type Dithiolate Ligands, Bis(tetrathiafulvalenedithiolato)palladium Determined by Powder X-ray Diffraction
Authors: Suzuki Wakako , Fujiwara Emiko , Kobayashi Akiko , Fujishiro Yuichi , Nishibori Eiji , Takata Masaki , Sakata Makoto , Okano Yoshinori , Kobayashi Hayao
Published: November 2003

A new palladium complex with extended-TTF (TTF = tetrathiafulvalene) dithiolate ligands, [Pd(dt)2] (dt = tetrathiafulvalenedithiolate) was prepared and its crystal structure was determined by synchrotron radiation X-ray powder experiments. [Pd(dt)2] is a semiconductor with a room temperature conductivity of 0.3 S cm-1.
Time-resolved Chemiluminescence Study of Photocatalytic Reaction of TiO2
Authors: Wu Xing-Zheng , Akiyama Keiko
Published: November 2003

Time-resolved luminol chemiluminescence (CL) with a time resolution of μs in photocatalytic reaction of TiO2 suspension was reported. It was found that the luminol CL signal was observed after about 15 μs of illumination of a pulse light of 355 nm, and reached maximum at about 40–60 μs, then decreased slowly with time.
Strategic Marketing Planning for Radically New Products
Authors: Cooper Lee G.
Published: November 2003

In this article, the author outlines an approach to marketing planning for radically new products, disruptive or discontinuous innovations that change the dimensionality of the consumer decision. The planning process begins with an extensive situation analysis. The factors identified in the situation analysis are woven into the economic webs surrounding the new product. The webs are mapped into Bayesian networks that can be updated as events unfold and used to simulate the impact that changes in assumptions underlying the web have on the prospects for the new product. The author illustrates this method using a historical case regarding the introduction of videotape recorders by Sony and JVC and a contemporary case of the introduction of electric vehicles. The author provides a complete, numerical example pertaining to a software development project in the Appendix.
An Examination of Organizational Factors Influencing New Product Success in Internal and Alliance-Based Processes
Authors: Sivadas Eugene , Dwyer F. Robert
Published: November 2003

New products provide increased sales, profits, and competitive strength for most organizations. However, nearly 50% of the new products that are introduced each year fail. Organizations thus find themselves in a double bind. On the one hand they must innovate consistently to remain competitive, but on the other hand innovation is risky and expensive. Many organizations are forming business alliances to quicken the pace of and reduce risks associated with innovation. Yet by some estimates, 70% of these alliances fail. Many of the prescriptions for successful alliance management clash with recommendations for effective innovation management. The authors develop testable hypotheses by integrating the new products and alliance literature. A construct—cooperative competency—derived from related concepts of mutual adjustment, absorptive capacity, and relational capability is posited as the key factor affecting new product development success, regardless of whether it is an intra- or interfirm endeavor. The authors test the model with data from a sample survey in the semiconductor manufacturing context and replicate it in the health care sector. The antecedents of cooperative competency—formalized and clannish administration, mutual dependence, and institutional support—are revealed empirically and substantiated. The authors identify the importance and means of developing interfirm cooperation.
Antecedents and Consequences of Marketing Managers’ Conflict-Handling Behaviors
Authors: Song X. Michael , Xie Jinhong , Dyer Barbara
Published: November 2003

In this study, the authors develop and test a cross-cultural conceptual model of the organizational-based antecedents and consequences of marketing managers’ conflict-handling behaviors during the new product development process. The hypotheses were tested using data collected from 968 companies in Japan, China (Hong Kong), the United States, and the United Kingdom. The results provide some interesting insights regarding the varying role of conflict management among diverse cultures.