264 research outputs found
Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion
No full textCharacterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function
An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis
No full textFenestrae are small pores in the endothelium of renal glomerular, gastrointestinal, and endocrine gland capillaries and are involved in the bidirectional exchange of molecules between blood and tissues. Although decades of studies have characterized fenestrae at the ultrastructural level, little is known on the mechanisms by which fenestrae form. We present the development of an in vitro assay in which rapid and abundant fenestra induction enables a detailed study of their biogenesis. Through the use of agents that stabilize or disassemble actin microfilaments, we show that actin microfilament remodeling is part of fenestra biogenesis in this model. Furthermore, by using a loss-of-function approach, we show that the diaphragm protein PV-1 is necessary for fenestral pore architecture and the ordered arrangement of fenestrae in sieve plates. Together, these data provide insight into the cell biology of fenestra formation and open up the future study of the fenestra to a combined morphological and biochemical analysis
Delta-like 4 is indispensable in thymic environment specific for T cell development.
No full textThe thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells
Analysis of the transcriptional program of developing induced regulatory T cells.
No full textCD25+ regulatory T cells develop in the thymus (nTregs), but may also be generated in the periphery upon stimulation of naive CD4 T cells under appropriate conditions (iTregs). To gain insight into the mechanisms governing iTreg development, we performed longitudinal transcriptional profiling of CD25+ T cells during their differentiation from uncommitted naive CD4 T cells. Microarray analysis of mRNA from CD25+ iTregs early after stimulation revealed expression of genes involved in cell cycle progression and T cell activation, which largely overlapped with genes expressed in CD25+ effector T cells (Teffs) used as a control. Whereas expression of these genes remained elevated in Teffs, it declined gradually in developing iTregs, resulting in a more quiescent phenotype in mature iTregs. A similar pattern of kinetics was observed for biological processes and for intracellular pathways over-represented within the expressed genes. A maximum dichotomy of transcriptional activity between iTregs and Teffs was reached at late stages of their maturation. Of interest, members of the FoxO and FoxM1 transcription factor family pathways exhibited a reciprocal expression pattern in iTregs and Teffs, suggesting a role of these transcription factors in determining T cell fate
Vascular endothelial growth factor restores delayed tumor progression in tumors depleted of macrophages
No full textGenetic depletion of macrophages in Polyoma Middle T oncoprotein (PyMT)-induced mammary tumors in mice delayed the angiogenic switch and the progression to malignancy. To determine whether vascular endothelial growth factor A (VEGF-A) produced by tumor-associated macrophages regulated the onset of the angiogenic switch, a genetic approach was used to restore expression of VEGF-A into tumors at the benign stages. This stimulated formation of a high-density vessel network and in macrophage-depleted mice, was followed by accelerated tumor progression. The expression of VEGF-A led to a massive infiltration into the tumor of leukocytes that were mostly macrophages. This study suggests that macrophage-produced VEGF regulates malignant progression through stimulating tumor angiogenesis, leukocytic infiltration and tumor cell invasion
An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic spindle
No full text12 pages, 8 figures.During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1-3-micron fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.D.T. Shima is a Hitchings-Elion fellow supported by the Burroughs
Wellcome Fund and N. Cabrera-Poch is a recipient of a Postdoctoral Fellowship
from the Spanish Ministry of Education and Culture.Peer reviewe
Detection of superparamagnetic beads with TMR sensors
No full textWalkenhorst S. Detection of superparamagnetic beads with TMR sensors. Bielefeld (Germany): Bielefeld University; 2007.Bislang war eine Signalverstärkung nötig für die Detektion einzelner magnetischer Marker mittels magnetoresistiver Sensoren. Die Detektion ohne Signalverstärkung mit Hilfe von magnetischen Tunnelelementen (englisch: "magnetic tunnel junction", MTJ) wäre ein Fortschritt im Bereich der Miniaturisierung und in der Anwendbarkeit als Biosensoren.
Versuche mit konventionellen AlOx-Tunnelelementen wurden durchgeführt in Verbindung mit superparamagnetischen Teilchen, sog. Beads, die senkrecht zur Sensorebene gesättigt wurden. TMR-Messungen von Sensoren mit und ohne Bead, gesättigt und ungesättigt, wurden miteinander verglichen. Mit Helmholtz-Spulen ist eine verschobene Hysterese möglich, die auch schmaler ist, dagegen scheinen die Aufbauten mit Permanentmagneten nicht einsetzbar zu sein, da das magnetische Feld nicht homogen genug war.
Von Elementen mit senkrechtem "pinning" wird erwartet, dass sie keine Hysterese zeigen. Für MgO-Tunnelelemente wurde senkrechtes pinning durch Formanisotropie verwirklicht. Mittels Elektronenstrahllithografie und Ionenätzen wurden Felder mit tausenden (Sub-)Mikrometerelementen hergestellt. Messungen auf Grundlage des magnetooptischen Kerr-Effekts zeigen Hysterese-freie Signale, wenn mit kleinen Feldern gemessen wird.Signal enhancement was so far necessary for detection of single magnetic markers using magnetoresistive sensors. Detection without signal enhancement on basis of a magnetic tunnel junction (MTJ) would be an important feature for miniaturization and for applications such as biosensors.
Based on conventional MTJs with AlOx barrier several setups with out-of-plane saturation of superparamagnetic beads were analyzed. TMR measurements with and without beads on top of a sensor, alternatively saturated or not were compared. With Helmholtz coils a shifted and narrow hysteresis is possible, whereas permanent magnets seem to be not applicable due to a very inhomogeneous field in this setup.
Elements are expected to have a hysteresis-free magnetic switching due to a perpendicular pinning. For MgO based sensors perpendicular pinning has been established via shape anisotropy. Arrays containing thousands of (sub-)micron elements were structured by electron beam lithography and ion beam etching. Measurements using the magneto-optical Kerr effect show a hysteresis-free signal for low magnetic fields
Qualitative Analysis of Interviews with Municipal Officers Toward the Human-Centered Improvement of the eLen Regulation Database System
No full textPart 1: Ethical and Legal Considerations in a Data-Driven SocietyInternational audienceThe objective of this study is to analyze results of interviews that we conducted with Japanese municipal officials who have engaged in legislative drafting and to present main issues addressed by the interviewees. Moreover, based on the results of analysis, it aims to clarify remaining problems that legislators face with during legislation, which will be necessary conditions for expanding and improving human-centered functions of e-legislation systems. Using qualitative analysis of interviews with municipal officers, this paper identifies the following four issues addressed by interviewees: (1) inconsistency among ordinances; (2) inconsistency of an ordinance; (3) insufficiency in consideration of legislative objectives and facts; (4) inadequacy of legal research. Based on the results of interview analysis, this study clarifies whether the eLen regulation database system copes with them and discusses remaining problems. Overall, it illustrates that some functions included in the eLen are helpful for diminishing those issues. However, in order to overcome the problems with which legislators face in the process of legislation, the results of this study show that it is significant to provide legislators education, such as trainings for the way to use the system and benchmarking method or for learning legislation process. Although the eLen has already implemented several instructive mechanisms, we will improve further the system so that users can learn the proper process of legislation through the usage of the system
Novel scaling behavior of the Ising model on curved surfaces
No full textWe demonstrate the nontrivial scaling behavior of Ising models defined on (i) a donut-shaped surface and (ii) a curved surface with a constant negative curvature. By performing Monte Carlo simulations, we find that the former model has two distinct critical temperatures at which both the specific heat C(T) and magnetic susceptibility chi(T) show sharp peaks. The critical exponents associated with the two critical temperatures are evaluated by the finite-size scaling analysis; the result reveals that the values of these exponents vary depending on the temperature range under consideration. In the case of the latter model, it is found that static and dynamic critical exponents deviate from those of the Ising model on a flat plane; this is a direct consequence of the constant negative curvature of the underlying surface
[[alternative]]Study on the genetic variation of Trochodendron aralioides and its phylogenetic relationship with allies.
No full text[[abstract]]Trochodendron aralioides Sieb. & Zucc., a species of the monotypic family Trochodendraceae is an evergreen tree characterized by vesselless wood. This primitive angiosperm is restricted to the East Asia in distribution. The purpose of this study is to investigate the population structure of this narrow distributed species and to explore the phylogeny of T. aralioides and its allies.
Multiple data of allozyme, and nucleotide sequences of the internal transcribed spacers (ITS) of ribosomal DNA were used to investigate genetic structure. Nineteen (allozyme) and twenty (ITS) populations were analyzed, including 16 from Taiwan, four from the main islands of Japan, Iriomote Island and Amami-O-shima. Genetic variability at the species level was comparable to that of narrow distributed species. In comparisons of allozyme, and ITS DNA sequences, the following results are concluded: first, genetic diversity was largely distributed within, rather than among, populations. Plants with protogynous and protandrous sexuality are highly self-incompatible, which probably promotes outcrossing and contributes to the high levels of heterozygosity within populations. Second, populations of T. aralioides were grouped into two distinct clusters, i.e., Japan/Amami-O-shima and Taiwan/Iriomote Island, indicating historical division undergone genetic differentiation. Populations of Iriomote Island was closely related to northern Taiwan. Those of glacial history of this area. Third, based on AMOVA analysis and no share an ITS haplotype, suggesting that geographic barriers have impeded gene flow. The genetic differentiation maybe due to geographic vicariance. Fourth, the isolation by distance model does not fit populations in Taiwan, but suitably describes the relationship between populations of T. aralioides in Japan and Taiwan.
In the second part, the phylogeny of representative species from seven families of the lower Hamamelidae was examined based on DNA sequences of 5.8S rDNA, trnL (UAA) intron and rbcL-atpb intergenic spacer. Three genera of the higher Hamamelidae, Juglans, Carpinus and Daphniphyllum were included. Cladistic analyses suggested paraphyly of the lower Hamamelidae, rooted at Michelia of Magnoliaceae. The 50% majority-rule consensus most parsimonious tree, maximum-likelihood tree and neighbor-joining tree were reconstructed using PAUP based on combined datas. All of the parsimonious trees of 1098 steps, with a CI and an RI of 0.837 and 0.691, respectively, were recovered. All reconstructed trees suggested that the lower Hamamelidae are a paraphyletic group. The Cercidiphyllaceae are most closely related to Hamamelidaceae, suggesting that Cercidiphyllaceae be placed in the Hamamelidales. Trochodendraceae and Tetracentraceae are very close as indicated by a bootstrap value of 100%. The Eupteleaceae phylogenetially are closely related with the Platanaceae.
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