bluuebunny/crossref_metadata_2025 huggingface dataset:
Topology optimization of slightly compressible fluids
Authors: Evgrafov A.
Published: July 2005

We consider the problem of optimal design of flow domains for Navier–Stokes flows in order to minimize a given performance functional. We attack the problem using topology optimization techniques, or control in coefficients, which are widely known in structural optimization of solid structures for their flexibility, generality, and yet ease of use and integration with existing FEM software. Topology optimization rapidly finds its way into other areas of optimal design, yet until recently it has not been applied to problems in fluid mechanics. The success of topology optimization methods for the minimal drag design of domains for Stokes fluids (see the study of Borrvall and Petersson [12]) has lead to attempts to use the same optimization model for designing domains for incompressible Navier–Stokes flows. We show that the optimal control problem obtained as a result of such a straightforward generalization is ill‐posed, at least if attacked by the direct method of calculus of variations. We illustrate the two key difficulties with simple numerical examples and propose changes in the optimization model that allow us to overcome these difficulties. Namely, to deal with impenetrable inner walls that may appear in the flow domain we slightly relax the incompressibility constraint as typically done in penalty methods for solving the incompressible Navier–Stokes equations. In addition, to prevent discontinuous changes in the flow due to very small impenetrable parts of the domain that may disappear, we consider so‐called filtered designs, that has become a “classic” tool in the topology optimization toolbox. Technically, however, our use of filters differs significantly from their use in the structural optimization problems in solid mechanics, owing to the very unlike design parametrizations in the two models. We rigorously establish the well‐posedness of the proposed model and then discuss related computational issues.
A numerical study on the suppression of draw resonance by inertia
Authors: Hagen T. , Langwallner B.
Published: July 2005

We study the one‐dimensional equations governing the formation of viscous fluid fibers in forced elongation when inertia enters the dominant balance. Even though it has been known for a long time that quite weak inertial forces suffice to suppress the onset of draw resonance, we shall give quantitatively more precise results than those reported before in the context of linear stability analysis. In addition, we shall demonstrate by numerical simulation that these findings remain valid in the nonlinear regime.
Jane Avril and the Entertainment Lithograph: The Female Celebrity and fin-de-siècle Questions of Corporeality and Performance
Authors: PEDLEY-HINDSON CATHERINE
Published: July 2005

Celebrity status at the fin de siècle depended largely on the creation and sustenation of a high profile, easily recogniZable image and the entertainment lithograph offered the most powerful and culturally pervasive means to achieve this. Thanks to the poster collectors of the period, and the acceptance of this ephemeral form into the world of ‘high art’, many examples of advertising imagery exist, offering a visual record of the themes and the performers that dominated the popular stage. This article focuses on poster works created by Jules Chéret (1836–1933) and Henri de Toulouse-Lautrec's (1864–1901) images of the dancer and actress Jane Avril, to explore whether the entertainment lithograph can be employed to aid an understanding and contextualization of fin-de-siècle popular performance.
Performing the City
Authors: MAKEHAM PAUL
Published: July 2005

Lewis Mumford, writing in the 1930s, understood the city as a ‘theater of social action’. Mumford's ideas remain important in the context of the contemporary post-industrial city, in which theatricality and performativity are key drivers of so-called ‘experience economies’. Increasingly, urban planners are attuned to such theatrical notions as the ‘urban scene’ and ‘urban drama’ in framing policy. Adopting interpretive strategies enabled by Performance Studies, this paper gives an account of some of the ways in which theatre and performance are made manifest in cities. It considers some of the implications of urban performativity, arguing that good city planning demands an ethics of performance, whereby citizens become spectators and co-performers in the urban drama.
Neuroprotective Effect of Recombinant Human Granulocyte Colony-Stimulating Factor in Transient Focal Ischemia of Mice
Authors: Komine-Kobayashi Miki , Zhang Ning , Liu Meizi , Tanaka Ryota , Hara Hideaki , Osaka Akimichi , Mochizuki Hideki , Mizuno Yoshikuni , Urabe Takao
Published: July 2005

Cerebral ischemia induces the expression of several growth factors and cytokines, which protect neurons against ischemic insults. Recent studies showed that granulocyte colony-stimulating factor (G-CSF) has a neuroprotective effect through the signaling pathway for the antiapoptotic cascade. The current study was designed to assess the neuroprotective mechanisms of G-CSF in ischemia/reperfusion injury using bone marrow chimera mice known to express enhanced green fluorescent protein (EGFP). Mice were subjected to ischemia/reperfusion and divided into two groups: those treated with G-CSF (G-CSF group) and vehicle (control group) ( n = 35 in each group). Immunohistochemistry and immunoblotting for antiapoptotic protein, nitrotyrosine, and inducible nitrate oxide synthase (iNOS) were performed. G-CSF significantly reduced stroke volume (34%, P < 0.006). G-CSF upregulated Stat3, pStat3, and Bcl-2 ( P < 0.05), and suppressed iNOS and nitrotyrosine expression. In EGFP chimera mice, G-CSF decreased the migration of lba-1/EGFP-positive bone marrow-derived monocytes/macrophages and increased intrinsic microglia/macrophages at ischemic penumbra ( P < 0.05), suggesting that bone marrow-derived monocytes/macrophages are not involved in G-CSF-induced reduction of ischemic injury size. Our study indicated that G-CSF exerts a neuroprotective effect through the direct activation of antiapoptotic pathway, and suggested that G-CSF is important for expansion of the therapeutic time window in patients with cerebral ischemia.
Neuroprotective Effect of Selective Kappa Opioid Receptor Agonist is Gender Specific and Linked to Reduced Neuronal Nitric Oxide
Authors: Zeynalov Emil , Nemoto Masaaki , Hurn Patricia D , Koehler Raymond C , Bhardwaj Anish
Published: July 2005

We have previously shown that treatment with selective kappa-opioid receptor agonist BRL 52537 hydrochloride [(±)-1-(3,4-dichlorophenyl) acetyl-2-(1-pyrrolidinyl) methylpiperidine] (1) has a long therapeutic window for providing ischemic neuroprotection and (2) attenuates ischemia-evoked nitric oxide (NO) production in vivo in rats. Neuronally derived NO has been shown to be deleterious in the male, but not in the female, rodent model of focal ischemic stroke. We sought to determine if the agent fails to protect ischemic brain when neuronal NO synthase (nNOS) is genetically deleted in male, but not female, mice. Halothane-anesthetized adult male and female nNOS null mutants (nNOS−/-) and the genetically matched wildtype (WT) strain were subjected to transient (2 h) middle cerebral artery occlusion by the intraluminal filament technique. Vehicle or BRL 52537 treatment with continuous intravenous infusion was instituted at the onset of reperfusion and continued for 22 h. In WT male mice, infarct volumes measured at 72 h of reperfusion were robustly decreased with BRL 52537 treatment. In contrast, BRL 52537 did not decrease infarct volume in male nNOS−/- mice. BRL 52537 had no effect in the WT or nNOS−/- female mice. These data support that BRL 52537% mechanism of neuroprotection in vivo is through attenuation of nNOS activity and ischemia-evoked NO production. Neuroprotective effects of BRL 52537 are lost in the male when nNOS is not present; therefore, BRL 52537 likely acts upstream from NO generation and its subsequent neurotoxicity.
IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK
Authors: Starkman Bela G. , Cravero John D. , Delcarlo Marcello , Loeser Richard F.
Published: July 2005

The IGF-I (insulin-like growth factor-I) signalling pathway responsible for regulation of proteoglycan synthesis in chondrocytes has not been defined and is the focus of the present study. Chondrocytes isolated from normal human articular cartilage were stimulated with IGF-I in monolayer culture or in suspension in alginate. IGF-I activated members of both the PI3K (phosphoinositide 3-kinase) pathway and the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway. The PI3K inhibitors LY294002 and wortmannin blocked IGF-I-stimulated Akt phosphorylation without blocking ERK phosphorylation and this was associated with complete inhibition of proteoglycan synthesis. A decrease in IGF-I-stimulated proteoglycan synthesis was also observed upon inhibition of mTOR (mammalian target of rapamycin) and p70S6 kinase, both of which are downstream of Akt. The MEK (MAPK/ERK kinase) inhibitors PD98059 and U0126 blocked IGF-I-stimulated ERK phosphorylation but did not block the phosphorylation of Akt and did not decrease proteoglycan synthesis. Instead, in alginate- cultured chondrocytes, the MEK inhibitors increased IGF-I-stimulated proteoglycan synthesis when compared with cells treated with IGF-I alone. This is the first study to demonstrate that IGF-I stimulation of the PI3K signalling pathway is responsible for the ability of IGF-I to increase proteoglycan synthesis. Although IGF-I also activates the ERK/MAPK pathway, ERK activity is not required for proteoglycan synthesis and may serve as a negative regulator.
Structure–function relationship in annexin A13, the founder member of the vertebrate family of annexins
Authors: Turnay Javier , Lecona Emilio , Fernández-Lizarbe Sara , Guzmán-Aránguez Ana , Fernández María Pilar , Olmo Nieves , Lizarbe Ma Antonia
Published: July 2005

Annexin A13 is considered the original progenitor of the 11 other members of vertebrate annexins, a superfamily of calcium/phospholipid-binding proteins. It is highly tissue-specific, being expressed only in intestinal and kidney epithelial cells. Alternative splicing generates two isoforms, both of which bind to rafts. In view of the lack of structural information supporting the physiological role of this annexin subfamily, we have cloned, expressed and purified human annexin A13b to investigate its structural and functional properties. The N-terminus of annexin A13b: (i) destabilizes the conserved protein core, as deduced from the low melting temperature in the absence (44 °C) or presence of calcium (55 °C), and (ii) impairs calcium-dependent binding to acidic phospholipids, requiring calcium concentrations >400 μM. Truncation of the N-terminus restores thermal stability and decreases the calcium requirement for phospholipid binding, confirming its essential role in the structure–function relationship of this annexin. Non-myristoylated annexin A13b only binds to acidic phospholipids at high calcium concentrations. We show for the first time that myristoylation of annexin A13b enables the direct binding to phosphatidylcholine, raft-like liposomes and acidic phospholipids in a calcium-independent manner. The conformational switch induced by calcium binding, from a ‘closed’ to an ‘open’ conformation with exposure of Trp227, can be mimicked by a decrease in pH, a process that may be relevant for membrane interactions. Our studies confirm that the common structural and functional characteristics that are dependent on the protein core of vertebrate annexins are likely to be common conserved features, whereas their variable N-termini confer distinct functional properties on annexins, as we report for myristoylation of annexin A13b.
Molecular action of 1,25-dihydroxyvitamin D3 and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site
Authors: Nutchey Barbara K. , Kaplan Josef S. , Dwivedi Prem P. , Omdahl John L. , Ferrante Antonio , May Brian K. , Hii Charles S. T.
Published: July 2005

Although investigations of the transcriptional regulation of the rat cytochrome P450C24 [CYP24 (25-hydroxyvitamin D3 24-hydroxylase)] gene by 1,25D (1,25-dihydroxyvitamin D3) at either the genomic, or more recently at the non-genomic, level have provided insight into the mechanism of control of 1,25D levels, this regulation is still poorly characterized. Using HEK-293T cells (human embryonic kidney 293T cells), we reported that 1,25D induction of CYP24 requires JNK (c-Jun N-terminal kinase) but not the ERK1/2 (extracellular-signal-regulated kinase 1/2). The phenomenon of synergistic up-regulation of CYP24 expression by PMA and 1,25D is well known and was found to be protein kinase C-dependent. Whereas ERK1/2 was not activated by 1,25D alone, its activation by PMA was potentiated by 1,25D also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of a dominant-negative ERK1(K71R) mutant (where K71R stands for Lys71→Arg), which resulted in a reduced level of synergy on a CYP24 promoter-luciferase construct. JNK was also shown to be required for synergy. We report, in the present study, the identification of a site located at −171/−163, about 30 bp upstream of the vitamin D response element-1 in the CYP24 proximal promoter. This sequence, 5′-TGTCGGTCA-3′, is critical for 1,25D induction of CYP24 and is therefore termed the vitamin D stimulatory element. The vitamin D stimulatory element, a target for the JNK module, and an Ets-1 binding site were shown to be vital for synergy between PMA and 1,25D. This is the first report to identify the DNA binding sequences required for the synergy between PMA and 1,25D and a role for JNK on the CYP24 gene promoter.
Mouse behavioural analysis in systems biology
Authors: van Meer Peter , Raber Jacob
Published: July 2005

Molecular techniques allowing in vivo modulation of gene expression have provided unique opportunities and challenges for behavioural studies aimed at understanding the function of particular genes or biological systems under physiological or pathological conditions. Although various animal models are available, the laboratory mouse (Mus musculus) has unique features and is therefore a preferred animal model. The mouse shares a remarkable genetic resemblance and aspects of behaviour with humans. In this review, first we describe common mouse models for behavioural analyses. As both genetic and environmental factors influence behavioural performance and need to be carefully evaluated in behavioural experiments, considerations for designing and interpretations of these experiments are subsequently discussed. Finally, common behavioural tests used to assess brain function are reviewed, and it is illustrated how behavioural tests are used to increase our understanding of the role of histaminergic neurotransmission in brain function.
The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism
Authors: Destoumieux-Garzón Delphine , Duquesne Sophie , Peduzzi Jean , Goulard Christophe , Desmadril Michel , Letellier Lucienne , Rebuffat Sylvie , Boulanger Pascale
Published: July 2005

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB–ExbB–ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA− strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a Kd of 1.2 μM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand–receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11–Pro16 β-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.
Secretion of cryptococcal phospholipase B1 (PLB1) is regulated by a glycosylphosphatidylinositol (GPI) anchor
Authors: Djordjevic Julianne T. , Del Poeta Maurizio , Sorrell Tania C. , Turner Kylie M. , Wright Lesley C.
Published: July 2005

The secreted, multifunctional enzyme PLB1 (phospholipase B1 protein encoded by the PLB1 gene) is a virulence determinant of the pathogenic fungus Cryptococcus neoformans, but the mechanism of its secretion is unknown. The cryptococcal PLB1 gene encodes putative, N-terminal LP (leader peptide) and C-terminal GPI (glycosylphosphatidylinositol) anchor attachment motifs, suggesting that PLB1 is GPI-anchored before secretion. To investigate the role of these motifs in PLB1 secretion, four cDNA constructs were created encoding the full-length construct (PLB1) and three truncated versions without the LP and/or the GPI anchor attachment motifs [LP−PLB1 (PLB1 expressed without the LP consensus motif), LP−PLB1GPI− (PLB1 expressed without the LP and GPI consensus motifs) and PLB1GPI− (PLB1 expressed without the GPI anchor attachment motif) respectively]. The constructs were ligated into pYES2, and galactose-induced expression was achieved in Saccharomyces cerevisiae. The LP was essential for secretion of the PLB1 protein and its three activities (PLB, lysophospholipase and lysophospholipase transacylase). Deletion of the GPI motif to create PLB1GPI− resulted in a redistribution of activity from the cell wall and membranes to the secreted and cytosolic fractions, with 36–54% of the total activity being secreted as compared with <5% for PLB1. PLB1 produced the maximum cell-associated activity (>2-fold more than that for PLB1GPI−), with 75–86% of this in the cell-wall fraction, 6–19% in the membrane fraction and 3–7% in the cytosolic fraction. Cell-wall localization was confirmed by release of activity with β-glucanase in both S. cerevisiae recombinants and wild-type C. neoformans. The dominant location of PLB1 in the cell wall via GPI anchoring may permit immediate release of the enzyme in response to changing environmental conditions and may represent part of a novel mechanism for regulating the secretion of a fungal virulence determinant.
Characterization of mouse amino acid transporter B0AT1 (slc6a19)
Authors: Böhmer Christoph , Bröer Angelika , Munzinger Michael , Kowalczuk Sonja , Rasko John E. J. , Lang Florian , Bröer Stefan
Published: July 2005

The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+–amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+–substrate–transporter] complex, which is consistent with the experimental data.
Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity
Authors: Furutani Yutaka , Manabe Ri-ichiroh , Tsutsui Ko , Yamada Tomiko , Sugimoto Nagisa , Fukuda Shiro , Kawai Jun , Sugiura Nobuo , Kimata Koji , Hayashizaki Yoshihide , Sekiguchi Kiyotoshi
Published: July 2005

We screened more than 60000 RIKEN mouse cDNAs for novel ECM (extracellular matrix) proteins by extensive computational screening followed by recombinant expression and immunohistochemical characterization. We identified two novel olfactomedin-family proteins characterized by the presence of tandem CXCXCX9C motifs in the N-terminal region, a coiled-coil domain and an olfactomedin domain in the C-terminal region. These proteins, named photomedin-1 and photomedin-2, were secreted as disulphide-bonded dimers (photomedin-1) or oligomers/multimers (photomedin-2) with O-linked carbohydrate chains, although photomedin-1 was proteolytically processed in the middle of the molecule after secretion. In the retina, photomedin-1 was selectively expressed in the outer segment of photoreceptor cells and photomedin-2 was expressed in all retinal neurons. Among a panel of ECM components, including glycosaminoglycans, photomedins preferentially bound to chondroitin sulphate-E and heparin. These results, together, indicate that photomedins are novel olfactomedin-domain-containing extracellular proteins capable of binding to proteoglycans containing these glycosaminoglycan chains.
Membrane binding of β2-glycoprotein I can be described by a two-state reaction model: an atomic force microscopy and surface plasmon resonance study
Authors: Gamsjaeger Roland , Johs Alexander , Gries Anna , Gruber Hermann J. , Romanin Christoph , Prassl Ruth , Hinterdorfer Peter
Published: July 2005

Complexes formed between β2GPI (β2-glycoprotein I), a human plasma protein, and biological membranes are considered to be targets of macrophages and antiphospholipid autoantibodies involved in autoimmune diseases, such as antiphospholipid syndrome or systemic lupus erythematosus. The positively charged lysine-rich fifth domain of β2GPI facilitates its interaction with phospholipid membranes containing acidic phospholipids, which normally become exposed by apoptotic processes. In the present study, atomic force microscopy was applied to visualize the binding of β2GPI to a mixed phospholipid model membrane at physiological ionic strength. On supported lipid bilayers the formation of supramolecular assemblies of the protein with a height of approx. 3.3 nm was observed, suggesting a lateral agglomeration of β2GPI. Detailed analysis of kinetic constants using surface plasmon resonance revealed that the binding can be described by a two-state reaction model, i.e. a very fast interaction step, depending on the content of acidic phospholipids in the bilayer, and a second step with significantly lower kon and koff values. Taken together, our results suggest a biphasic interaction mechanism: a fast step of β2GPI binding to negatively charged lipids, mainly based on electrostatic interactions, and a slower phase of agglomeration of the protein on the bilayer surface accompanied by a protein-induced rigidification of the membrane, as revealed by electron paramagnetic resonance.
Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene
Authors: Rentsendorj Otgonchimeg , Nagy Andrea , Sinkó Ildikó , Daraba Andreea , Barta Endre , Kiss Ibolya
Published: July 2005

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between −338 and +67. Histological analysis of the founder embryos demonstrated relatively weak transgene activity in the developing chondrocranium, axial and appendicular skeleton with highest level of expression in the columnar proliferating chondroblasts and prehypertrophic chondrocytes. Computer analysis of the matrilin-1 genes of amniotes revealed a highly conserved Pe1 (proximal promoter element 1) and two less-conserved sequence blocks in the distal promoter region. The inverted Sox motifs of the Pe1 element interacted with chondrogenic transcription factors Sox9, L-Sox5 and Sox6 in vitro and another factor bound to the spacer region. Point mutations in the Sox motifs or in the spacer region interfered with or altered the formation of nucleoprotein complexes in vitro and significantly decreased the reporter gene activity in transient expression assays in chondrocytes. In vivo occupancy of the Sox motifs in genomic footprinting in the expressing cell type, but not in fibroblasts, also supported the involvement of Pe1 in the tissue-specific regulation of the gene. Our results indicate that interaction of Pe1 with distal DNA elements is required for the high level, cartilage- and developmental stage-specific transgene expression.
Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts
Authors: Kotani Tomoya , Yamashita Masakane
Published: July 2005

Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-γ-tubulin antibody. The results indicate possible roles of γ-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.
Novel variants of human SCaMC-3, an isoform of the ATP-Mg/Pi mitochondrial carrier, generated by alternative splicing from 3′-flanking transposable elements
Authors: del Arco Araceli
Published: July 2005

CaMCs (calcium-dependent mitochondrial carriers) represent a novel subfamily of metabolite carriers of mitochondria. The ATP-Mg/Pi co-transporter, functionally characterized more than 20 years ago, has been identified to be a CaMC member. There are three isoforms of the ATP-Mg/Pi carrier in mammals, SCaMC-1 (short CaMC-1), -2 and -3 (or APC-1, -3 and -2 respectively), corresponding to the genes SLC25A24, SLC25A25 and SLC25A23 respectively, as well as six N-terminal variants generated by alternative splicing for SCaMC-1 and -2 isoforms. In the present study, we describe four new variants of human SCaMC-3 generated by alternative splicing. The new mRNAs use the exon 9 3′-donor site and distinct 5′-acceptor sites from repetitive elements, in regions downstream of exon 10, the last exon in all SCaMCs. Transcripts lacking exon 10 (SCaMC-3b, -3b′, -3c and -3d) code for shortened proteins lacking the last transmembrane domain of 422, 456 and 435 amino acids, and were found in human tissues and HEK-293T cells. Mitochondrial targeting of overexpressed SCaMC-3 variants is incomplete. Surprisingly, the import impairment is overcome by removing the N-terminal extension of these proteins, suggesting that the hydrophilic N-terminal domain also participates in the mitochondrial import process, as shown for the CaMC members aralar and citrin [Roesch, Hynds, Varga, Tranebjaerg and Koehler (2004) Hum. Mol. Genet. 13, 2101–2111].
Characterization of a novel Foxa (hepatocyte nuclear factor-3) site in the glucagon promoter that is conserved between rodents and humans
Authors: Sharma Sanjeev K. , Leinemann Ulrike , Ratke Regine , Oetjen Elke , Blume Roland , Dickel Corinna , Knepel Willhart
Published: July 2005

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the Foxa [Fox (forkhead box) subclass A; also known as HNF-3 (hepatocyte nuclear factor-3)] family are required for cell-specific activation of the glucagon gene in pancreatic islet α-cells. However, their action on the glucagon gene is poorly understood. In the present study, comparative sequence analysis and molecular characterization using protein–DNA binding and transient transfection assays revealed that the well-characterized Foxa-binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. A novel Foxa site was identified that is conserved in rats, mice and humans. It mediates activation of the glucagon gene by Foxa proteins and confers cell-specific promoter activity in glucagon-producing pancreatic islet α-cell lines. In contrast with previously identified Foxa-binding sites in the glucagon promoter, which bind nuclear Foxa2, the novel Foxa site was found to bind preferentially Foxa1 in nuclear extracts of a glucagon-producing pancreatic islet α-cell line, offering a mechanism that explains the decrease in glucagon gene expression in Foxa1-deficient mice. This site is located just upstream of the TATA box (between −30 and −50), suggesting a role for Foxa proteins in addition to direct transcriptional activation, such as a role in opening the chromatin at the start site of transcription of the glucagon gene.
Mobilization of the iron centre in IscA for the iron–sulphur cluster assembly in IscU
Authors: Ding Baojin , Smith Edward S. , Ding Huangen
Published: July 2005

The biogenesis of iron–sulphur clusters requires the co-ordinated delivery of both iron and sulphur. It is now clear that sulphur in iron–sulphur clusters is derived from L-cysteine by cysteine desulphurases. However, the iron donor for the iron–sulphur cluster assembly still remains elusive. Our previous studies indicated that Escherichia coli IscA, a member of the iron–sulphur cluster assembly machinery, is an iron-binding protein that can provide iron for the iron–sulphur cluster assembly in a proposed scaffold IscU. To determine how the iron centre in IscA is transferred for the iron–sulphur cluster assembly in IscU, we explore the mobility of the iron centre in IscA. The UV–visible and EPR measurements show that L-cysteine, but not IscU, is able to mobilize the iron centre in IscA and make the iron available for the iron–sulphur cluster assembly in IscU. Other related biological thiols such as N-acetyl-L-cysteine or reduced glutathione have no effect on the iron centre of IscA, suggesting that L-cysteine is unique in mobilizing the iron centre of IscA. Nevertheless, L-cysteine alone is not sufficient to transfer the iron from IscA to IscU. Both L-cysteine and cysteine desulphurase (IscS) are required for the IscA-mediated assembly of iron–sulphur clusters in IscU. The results suggest that L-cysteine may have two distinct functions in the biogenesis of iron–sulphur clusters: to mobilize the iron centre in IscA and to provide sulphur via cysteine desulphurase (IscS) for the iron–sulphur cluster assembly in IscU.
Regulation of ecdysteroid signalling during Drosophila development: identification, characterization and modelling of ecdysone oxidase, an enzyme involved in control of ligand concentration
Authors: Takeuchi Hajime , Rigden Daniel J. , Ebrahimi Bahram , Turner Philip C. , Rees Huw H.
Published: July 2005

The steroidal moulting hormones (ecdysteroids) mediate developmental transitions in insects, and their regulation is mainly controlled by the production and inactivation of these steroid hormones at the appropriate developmental times. One route of metabolism of ecdysteroids in insects involves EO (ecdysone oxidase)-catalysed conversion into 3-dehydroecdysteroid, which undergoes reduction to the corresponding 3-epiecdysteroid. By a twin-stranded bioinformatics approach, employing both phylogenomics and model structure-based analysis, we first predicted that DmEO (the EO of Drosophila melanogaster) corresponds to the protein product of gene CG9504. When CG9504 was expressed in COS7 cells, significant conversion of ecdysone into 3-dehydroecdysone was observed. Quantitative PCR and enzyme assay showed that DmEO was mainly expressed in the midgut during the late instars at a time corresponding to a hormone titre peak. DmEO shares only 27% amino acid sequence identity with Spodoptera littoralis (Lepidoptera) EO, yet key substrate-binding residues are well conserved. A model of DmEO is consistent with an inability to catalyse reaction of cholesterol derivatives. The significance of DmEO in ligand activation is discussed in relation to new evidence suggesting that 3-dehydro- and 3-epiecdysteroids may be functionally active as ligands in a novel, atypical ecdysteroid signalling pathway involving the Drosophila orphan nuclear receptor, DHR38, rather than being merely hormone inactivation products.
Identification of calcium-regulated heat-stable protein of 24 kDa (CRHSP24) as a physiological substrate for PKB and RSK using KESTREL
Authors: Auld Gillian C. , Campbell David G. , Morrice Nick , Cohen Philip
Published: July 2005

A substrate for PKBα (protein kinase Bα) was detected in liver extracts, and was purified and identified as CRHSP24 (calcium-regulated heat-stable protein of apparent molecular mass 24 kDa). PKBα, as well as SGK1 (serum- and glucocorticoid-induced protein kinase 1) and RSK (p90 ribosomal S6 kinase), phosphorylated CRHSP24 stoichiometrically at Ser52in vitro and its brain-specific isoform PIPPin at the equivalent residue (Ser58). CRHSP24 became phosphorylated at Ser52 when HEK-293 (human embryonic kidney) cells were stimulated with IGF-1 (insulin-like growth factor-1) and this was prevented by inhibitors of PI3K (phosphoinositide 3-kinase), but not by rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] or PD 184352, an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade and hence the activation of RSK. IGF-1 induced a similar phosphorylation of CRHSP24 in ES (embryonic stem) cells from wild-type mice or mice that express the PDK1 (3-phosphoinositide-dependent kinase 1) mutant (PDK1[L155E]) that activates PKBα normally, but cannot activate SGK. CRHSP24 also became phosphorylated at Ser52 in response to EGF (epidermal growth factor) and this was prevented by blocking activation of both the classical MAPK cascade and the activation of PKBα, but not if just one of these pathways was inhibited. DYRK2 (dual-specificity tyrosine-phosphorylated and -regulated protein kinase 2) phosphorylated CRHSP24 at Ser30, Ser32 and Ser41in vitro, and Ser41 was identified as a site phosphorylated in cells. These and other results demonstrate that CRHSP24 is phosphorylated at Ser52 by PKBα in response to IGF-1, at Ser52 by PKBα and RSK in response to EGF, and at Ser41 in the absence of IGF-1/EGF by a DYRK isoform or another proline-directed protein kinase(s).
Numerical study of liquid-hydrogen droplet generation from a vibrating orifice
Authors: Xu J. , Celik D. , Hussaini M. Y. , Van Sciver S. W.
Published: July 2005

Atomic hydrogen propellant feed systems for far-future spacecraft may utilize solid-hydrogen particle carriers for atomic species that undergo recombination to create hot rocket exhaust. Such technology will require the development of particle generation techniques. One such technique could involve the production of hydrogen droplets from a vibrating orifice that would then freeze in cryogenic helium vapor. Among other quantities, the shape and size of the droplet are of particular interest. The present paper addresses this problem within the framework of the incompressible Navier–Stokes equations for multiphase flows, in order to unravel the basic mechanisms of droplet formation with a view to control them. Surface tension, one of the most important mechanisms to determine droplet shape, is modeled as the source term in the momentum equation. Droplet shape is tracked using a volume-of-fluid approach. A dynamic meshing technique is employed to accommodate the vibration of the generator orifice. Numerically predicted droplet shapes show satisfactory agreement with photographs of droplets generated in experiments. A parametric study is carried out to understand the influence of injection velocity, nozzle vibrational frequency, and amplitude on the droplet shape and size. The computational model provides a definitive qualitative picture of the evolution of droplet shape as a function of the operating parameters. It is observed that, primarily, the orifice vibrational frequency affects the shape, the vibrational amplitude affects the time until droplet detachment from the orifice, and the injection velocity affects the size. However, it does not mean that, for example, there is no secondary effect of amplitude on shape or size.
Quasirelativistic theory for the magnetic shielding constant. III. Quasirelativistic second-order Møller–Plesset perturbation theory and its application to tellurium compounds
Authors: Fukuda Ryoichi , Nakatsuji Hiroshi
Published: July 2005

The quasirelativistic (QR) generalized unrestricted Hartree–Fock method for the magnetic shielding constant [R. Fukuda, M. Hada, and H. Nakatsuji, J. Chem. Phys. 118, 1015 (2003); R. Fukuda, M. Hada, and H. Nakatsuji, J. Chem. Phys.118, 1027 (2003)] has been extended to include the electron correlation effect in the level of the second-order Møller–Plesset perturbation theory (MP2). We have implemented the energy gradient and finite-perturbation methods to calculate the magnetic shielding constant at the QR MP2 level and applied to the magnetic shielding constants and the NMR chemical shifts of Te125 nucleus in various tellurium compounds. The calculated magnetic shielding constants and NMR chemical shifts well reproduced the experimental values. The relations of the chemical shifts with the natures of ligands, and the tellurium oxidation states were investigated. The chemical shifts in different valence states were explained by the paramagnetic shielding and spin-orbit terms. The tellurium 5p electrons are the dominant origin of the chemical shifts in the TeI and TeII compounds and the chemical shifts were explained by the p-hole mechanism. The tellurium d electrons also play an important role in the chemical shifts of the hypervalent compounds.
Molecular simulation of the shear viscosity and the self-diffusion coefficient of mercury along the vapor-liquid coexistence curve
Authors: Raabe Gabriele , Todd B. D. , Sadus Richard J.
Published: July 2005

In earlier work [G. Raabe and R. J. Sadus, J. Chem. Phys. 119, 6691 (2003)] we reported that the combination of an accurate two-body ab initio potential with an empirically determined multibody contribution enables the prediction of the phase coexistence properties, the heats of vaporization, and the pair distribution functions of mercury with reasonable accuracy. In this work we present molecular dynamics simulation results for the shear viscosity and self-diffusion coefficient of mercury along the vapor-liquid coexistence curve using our empirical effective potential. The comparison with experiment and calculations based on a modified Enskog theory shows that our multibody contribution yields reliable predictions of the self-diffusion coefficient at all densities. Good results are also obtained for the shear viscosity of mercury at low to moderate densities. Increasing deviations between the simulation and experimental viscosity data at high densities suggest that not only a temperature-dependent but also a density-dependent multibody contribution is necessary to account for the effect of intermolecular interactions in liquid metals. An analysis of our simulation data near the critical point yields a critical exponent of β=0.39, which is identical to the value obtained from the analysis of the experimental saturation densities.
Equilibrium solvation in quadrupolar solvents
Authors: Milischuk Anatoli A. , Matyushov Dmitry V.
Published: July 2005

We present a microscopic theory of equilibrium solvation in solvents with zero dipole moment and nonzero quadrupole moment (quadrupolar solvents). The theory is formulated in terms of autocorrelation functions of the quadrupolar polarization (structure factors). It can be therefore applied to an arbitrary dense quadrupolar solvent for which the structure factors are defined. We formulate a simple analytical perturbation treatment for the structure factors. The solute is described by coordinates, radii, and partial charges of constituent atoms. The theory is tested on Monte Carlo simulations of solvation in model quadrupolar solvents. It is also applied to the calculation of the activation barrier of electron transfer reactions in a cleft-shaped donor-bridge-acceptor complex dissolved in benzene with the structure factors of quadrupolar polarization obtained from molecular-dynamics simulations.
Unfaulting of dislocation loops in the GaInNAs alloy: An estimation of the stacking fault energy
Authors: Herrera M. , González D. , Lozano J. G. , García R. , Hopkinson M. , Liu H. Y. , Gutierrez M. , Navaretti P.
Published: July 2005

A study by transmission electron microscopy of the influence of the In and N contents in the ranges of 20%–35% and 1.1%–3%, respectively, on the microstructure of Ga1−xInxNyAs1−y quantum wells is presented. Frank dislocation loops characterized as extrinsic have been found in the samples with x⩾0.25. In these structures, threading dislocations appear as a consequence of the unfaulting of the loops for y⩾0.014. An analysis of the density and size of the dislocation loops has provided an estimation of the critical radius for the unfaulting process. A model for this critical radius of the unfaulting process of extrinsic Frank loops is proposed. From this model and experimental values of critical radius, an estimation of the stacking fault energy of the GaInNAs alloy has been made. We have found a reduction in the stacking fault energy of the GaInNAs alloys when increasing the N content from 1.4% to 2.3% in good agreement with the theoretical estimation of the stacking fault energies of zinc-blende GaN and InN.
Local symmetry breaking in PbxSr1−xTiO3 ceramics and composites studied by Raman spectroscopy
Authors: Jain M. , Yuzyuk Yu. I. , Katiyar R. S. , Somiya Y. , Bhalla A. S.
Published: July 2005

Local symmetry breaking in PbxSr1−xTiO3 (PST) solid solutions was revealed by Raman spectroscopy below and above the ferroelectric phase-transition temperature. In the paraelectric phase, local symmetry breaking associated with the polar clusters localized mostly at grain boundaries in ceramic samples leads to the activation of the polar modes far above the dielectric maximum. This effect was found remarkably enhanced in the PST:MgO composites due to the compositional inhomogeneity of the grain boundaries and the intergrain stresses in the composite. In the ferroelectric phase, the degeneracy of the E(TO1) soft mode was lifted due to translational symmetry breaking in the solid solutions with x=0.2 and 0.3, where the substitution of Sr by Pb replaces partially the ionic bonding between the A cations and the oxygens by a stronger covalent bonding.
Development of a geometrical evaluation apparatus for ultrahigh 100 GB optical disk masters
Authors: Kuwahara Masashi , Fons Paul , Tominaga Junji , Honma Katsunori , Egawa Akira , Miyatani Tatsuya , Nakajima Kunio , Abe Hidekazu , Tokumoto Hiroshi
Published: July 2005

We report on the development of a prototype system for the automatic characterization of the pit shape in optical master disks (OMDS) and its relationship with the corresponding optical readout signal. The system consists of two basic components: an optical disk drive tester (ODDT) which uses a laser beam pickup to convert physical pits on the master disk into electrical signals and an integrated atomic force microscope (AFM). In this system, the ODDT scans the OMD and records the positions of errors. Using these recorded positions, the integrated tapping-mode AFM unit then automatically positions the AFM head to the corresponding locations on the OMD and initiates a scan which serves to record topographical information of pit shape in the error regions. In an initial trial, the system was used to automatically detect and record topographical information on randomly written error patterns (1μm in length) recorded on a 12 cm diam optical disk. For each identified region, 50μm square AFM scans were then automatically recorded. The system thus allows the direct observation of the correlation between errors detected in the optical readout signal and the topology of the corresponding pit on the nanometer scale. As high reliability of the AFM unit is a prerequisite for automatic operation, a carbon nanotube based probe was developed and proved to be much more reliable than conventional Si based cantilevers.
Time-resolved spin filtering in semiconductor symmetric resonant barrier structures
Authors: Yu Leo , Voskoboynikov O.
Published: July 2005

Spin-dependent tunneling in semiconductor symmetric double barrier structures is studied theoretically. Our calculation is based on the effective one-band Hamiltonian and Dresselhaus spin-orbit coupling. We demonstrate that the ratio of the tunneling times of electrons with opposite spin orientations can vary over a few orders in magnitude. The large and tunable ratio of the tunneling times can serve as the basis in the development of all-semiconductor dynamic spin filters.
Linear interaction analysis applied to a mixture of two perfect gases
Authors: Griffond J.
Published: July 2005

The linear interaction analysis is applied to mixtures of two perfect gases. Differences of molar mass and constant volume specific heat between both gases are taken into account as two new wave families. Transfer functions across the shock wave from these waves to acoustic, shear or entropy waves are given. The approach is validated against numerical simulations of the interaction of concentration spots with a shock wave. The case of turbulence creation by a shock wave traveling in an inhomogeneous mixture is considered.