bluuebunny/crossref_metadata_2025 huggingface dataset:
Wetting and dissolution studies of fluoropolymers used in 157 nm photolithography applications
Authors: Markley T. J. , Marsella J. A. , Robertson E. A. , Parris G. E. , Zarkov Z. , Jakubek V. , Ober C. K.
Published: February 2004

Photolithography using the F2 excimer laser at 157 nm, a technology to bridge traditional optical lithography and next generation lithographies, promises to enable ultralarge scale integrated devices with sub-70 nm design rules. Chemically amplified resists based on fluoropolymers have previously been shown to be good candidates for 157 nm microlithography. In our research, hexafluoroisopropyl alcohol (HFIPA) groups have been incorporated into polymers to improve the base solubility and to increase the transparency needed for new photoresists at 157 nm. These new polymers have absorbance values at 157 nm ranging from 1.7 to 3.9 μm−1. The introduction of fluorine groups increases their hydrophobicity and makes these polymers more difficult to wet at the surface. We have studied the effect of fluorine content on hydrophobicity of fluorinated polymers by measuring contact angle data over short time intervals. The ability to combine fluoropolymer synthesis with extensive contact angle studies has proven to be valuable in understanding solubility characteristics of these resins. These studies indicate that many of the fluorinated polymers have a very hydrophobic surface. Lowering contact angles can help to achieve the desired base solubility. Wetting studies also indicate that addition of certain surfactants to 0.26 N aqueous tetramethylammonium hydroxide (TMAH) help to reduce the contact angle. In addition to contact angle studies, we have used a quartz crystal microbalance to determine the dissolution rate of polymers in an aqueous base. Preliminary dissolution studies indicate a correlation to contact angle findings. Aqueous base solutions of 0.26 N TMAH containing surfactants were found to significantly lower the contact angle of an HFIPA-containing polymer, and increase the dissolution rate by an order of magnitude.
Growth of strained Si and strained Ge heterostructures on relaxed Si1−xGex by ultrahigh vacuum chemical vapor deposition
Authors: Lee Minjoo L. , Pitera Arthur J. , Fitzgerald E. A.
Published: February 2004

Numerous applications require the growth of planar strained-layer heterostructures on relaxed Si1−xGex. After briefly reviewing these applications as well as the challenges in growing such heterostructures, we provide experimental examples illustrating the influence of lattice mismatch, growth temperature, and film composition on the morphology of thin strained layers in the Ge–Si alloy system. Procedures for growing strained Si and strained Ge single and double heterostructures via ultrahigh vacuum chemical vapor deposition are described in detail. We demonstrate planar growth of strained Ge layers with lattice mismatches as high as 2%, planar Si layers on any Si1−xGex lattice constant, and double heterostructures that are comprised of a strained Ge layer capped with strained Si. Notably, the techniques described here have already been applied to the fabrication of extremely high mobility p- and n-channel metal–oxide–semiconductor field-effect transistors and germanium-on-insulator substrates.
Wave-front errors of reference spherical waves in high-numerical aperture point diffraction interferometers
Authors: Sekine Yoshiyuki , Suzuki Akiyoshi , Hasegawa Masanobu , Ouchi Chidane , Hara Shinichi , Hasegawa Takayuki , Kuramoto Yoshiyuki , Kato Seima , Murakami Katsuhiko , Saito Jun , Ota Kazuya , Kondo Hiroyuki , Ishii Mikihiko , Kawakami Jun , Oshino Tetsuya , Sugisaki Katsumi , Zhu Yucong , Otaki Katsura , Liu Zhiqiang
Published: February 2004

In phase-shifting point diffraction interferometry (PS/PDI), a pinhole with a diameter of 34 nm is necessary to measure a wave-front aberration of extreme ultraviolet projection optics of numerical aperture (NA) 0.20. However, it is extremely difficult to process such a small pinhole, and light transmission through the pinhole becomes too low. Here, the diameter of a pinhole is optimized, together with the thickness of a Ta membrane, for a converging wave of NA 0.20 with no aberration so that the difference between a wave front produced by the pinhole and that of a spherical wave is minimized. On that condition, the optimum values are a diameter of 50 nm and a thickness of 200 nm. For these values, behaviors are examined in real cases, including focal point shifts and aberrations with incident light. Astigmatism in the aberrations has the most impact on a wave-front error, and a 0°–90° astigmatism (Z5) coefficient in the FRINGE Zernike polynomials of test optics is required to be less than 50 mλ to use the pinhole in PS/PDI. For optics with a higher NA (∼0.25), the use of a Ta membrane is not adequate because of poor performance in small pinholes, and materials that have higher extinction coefficients are preferable.
Microfabrication of silicon–nitride micromesh bolometric detectors for planck high frequency instrument
Authors: Yun Minhee , Bock Jamie , Holmes Warren , Koch Tim , Mulder Jerry , Vasquez Richard P. , Wild Larry , Lange Andrew
Published: February 2004

The high frequency instrument (HFI) on the National Aeronautics and Space Administration/European Space Agency Planck Surveyor, scheduled for launch in 2007, will map the entire sky in six frequency bands ranging from 100 to 857 GHz to probe cosmic microwave background anisotropy and polarization with angular resolution ranging from 9′ to 5′. The HFI focal plane will contain 48 silicon–nitride micromesh bolometers operating from a 100 mK heat sink. Four detectors in each of the six bands will detect unpolarized radiation. An additional four pairs of detectors will provide sensitivity to linear polarization of emissions at 143, 217, and 353 GHz. We have fabricated and developed sensitive Si3N4 micromesh spider-web bolometers for submillimeter observation using microelectromechanical system techniques. The spiderweb architecture in this research provides high infrared absorption with minimal heat capacity and volume.
Chemical and electrical dopants profile evolution during solid phase epitaxial regrowth
Authors: Pawlak B. J. , Lindsay R. , Surdeanu R. , Dieu B. , Geenen L. , Hoflijk I. , Richard O. , Duffy R. , Clarysse T. , Brijs B. , Vandervorst W. , Dachs C. J. J.
Published: February 2004

Solid phase epitaxial regrowth (SPER) is a promising method for junction formation of sub-65 nm complementary metal–oxide–semiconductor technology nodes. This is mainly due to a high dopant activation level, easy control over electrical junction depth, excellent abruptness, and limited boron diffusion. In the present research we investigate in detail the activation process and the chemical profile change after SPER junction activation with respect to the regrowth temperature. We also obtain the electrically active profiles. We find that the process window for SPER between T=620 °C and T=740 °C offers the best activation level and has a dopant profile similar to the as-implanted. While increasing the regrowth temperature, we observe the gradual increase of the transient enhanced diffusion effect and formation of B trapping centers in the end-of-range (EOR) region. At temperatures as high as T=800 °C and T=850 °C the dopant activation beyond the original a-Si layer is observed and the high metastable B activation in the junction drops dramatically. All these changes can be associated with release of Si interstitials from the EOR region due to dissolution of 〈311〉 defects.
Mechanism of ArF resist-pattern shrinkage in critical-dimension scanning electron microscopy measurement
Authors: Azuma Tsukasa , Chiba Kenji , Abe Hideaki , Motoki Hiroshi , Sasaki Noriaki
Published: February 2004

ArF resist-pattern shrinkage in critical-dimension scanning electron microscopy (CD-SEM) measurement is investigated, and its mechanism is discussed. In CD-SEM irradiation damage in the resist detected by Fourier transform infrared (FTIR) spectroscopy, we propose that C=O units of pendant polymer chains decompose COOH units at the first stage and that the COOH units finally decompose CO2 outgas at the second stage. We propose the mechanism of the ArF resist-pattern shrinkage in the CD-SEM measurement from experimental and simulation results of the FTIR spectroscopy and Monte Carlo simulation of electron scattering trajectories into the resist. The first resist polymer free volume shrinkage is caused by hydrogen bonds between the COOH units, as well as decomposition of dissolution inhibitor groups. The second resist polymer free volume shrinkage is caused by the CO2 outgas within 4–14 nm in depth from the resist surface depending on the CD-SEM acceleration voltages of 400 and 800 V, respectively, with probe current of 8 pA. Based on the mechanism, we also propose an improvement in the ArF resist material design to reduce the ArF resist-pattern shrinkage in the CD-SEM measurement.
THE MOST GENERAL AND RENORMALIZABLE MAXIMAL ABELIAN GAUGE
Authors: SHINOHARA TORU , IMAI TAKAHITO , KONDO KEI-ICHI
Published: February 2004

We construct the most general gauge fixing and the associated Faddeev–Popov ghost term for the SU(2) Yang–Mills theory, which leaves the global U(1) gauge symmetry intact (i.e. the most general Maximal Abelian gauge). We show that the most general form involves eleven independent gauge parameters. Then we require various symmetries which help to reduce the number of independent parameters for obtaining the simpler form. In the simplest case, the off-diagonal part of the gauge fixing term obtained in this way is identical to the modified maximal Abelian gauge term with two gauge parameters which was proposed in the previous paper from the viewpoint of renormalizability. In this case, moreover, we calculate the beta function, anomalous dimensions of all fields and renormalization group functions of all gauge parameters in perturbation theory to one-loop order. We also discuss the implication of these results to obtain information on low-energy physics of QCD.
Features to consider in a data warehousing system
Authors: Gorla Narasimhaiah
Published: February 2004

Evaluating and assessing the important distinctions between data processing capability and data currency.
Measuring productivity in the software industry
Authors: Anselmo Donald , Ledgard Henry
Published: February 2004

Effective IT curricula balances tradition with innovation. One way to enhance that balance is to examine the common threads in the various knowledge areas.
Systems requirements for organizational learning
Authors: Chen Jim Q. , Lee Ted E. , Zhang Ruidong , Zhang Yue Jeff
Published: February 2004

To produce organizational memory, knowledge management systems must transform individual knowledge into organizational knowledge, sometimes grudgingly.
Tangible multimodal interfaces for safety-critical applications
Authors: Cohen Philip R. , McGee David R.
Published: February 2004

Designing effective digital systems in safety-critical arenas takes interfaces inspired by, or included in, the physical world.
Ontological user profiling in recommender systems
Authors: Middleton Stuart E. , Shadbolt Nigel R. , De Roure David C.
Published: February 2004

We explore a novel ontological approach to user profiling within recommender systems, working on the problem of recommending on-line academic research papers. Our two experimental systems, Quickstep and Foxtrot, create user profiles from unobtrusively monitored behaviour and relevance feedback, representing the profiles in terms of a research paper topic ontology. A novel profile visualization approach is taken to acquire profile feedback. Research papers are classified using ontological classes and collaborative recommendation algorithms used to recommend papers seen by similar people on their current topics of interest. Two small-scale experiments, with 24 subjects over 3 months, and a large-scale experiment, with 260 subjects over an academic year, are conducted to evaluate different aspects of our approach. Ontological inference is shown to improve user profiling, external ontological knowledge used to successfully bootstrap a recommender system and profile visualization employed to improve profiling accuracy. The overall performance of our ontological recommender systems are also presented and favourably compared to other systems in the literature.
Precongruence formats for decorated trace semantics
Authors: Bloom Bard , Fokkink Wan , van Glabbeek Rob J.
Published: February 2004

This paper explores the connection between semantic equivalences and preorders for concrete sequential processes, represented by means of labeled transition systems, and formats of transition system specifications using Plotkin's structural approach. For several preorders in the linear time---branching time spectrum a format is given, as general as possible, such that this preorder is a precongruence for all operators specifiable in that format. The formats are derived using the modal characterizations of the corresponding preorders.
Perceptually based brush strokes for nonphotorealistic visualization
Authors: Healey Christopher G. , Tateosian Laura , Enns James T. , Remple Mark
Published: February 2004

An important problem in the area of computer graphics is the visualization of large, complex information spaces. Datasets of this type have grown rapidly in recent years, both in number and in size. Images of the data stored in these collections must support rapid and accurate exploration and analysis. This article presents a method for constructing visualizations that are both effective and aesthetic. Our approach uses techniques from master paintings and human perception to visualize a multidimensional dataset. Individual data elements are drawn with one or more brush strokes that vary their appearance to represent the element's attribute values. The result is a
nonphotorealistic visualization
of information stored in the dataset. Our research extends existing glyph-based and nonphotorealistic techniques by applying perceptual guidelines to build an effective representation of the underlying data. The nonphotorealistic properties the strokes employ are selected from studies of the history and theory of Impressionist art. We show that these properties are similar to visual features that are detected by the low-level human visual system. This correspondence allows us to manage the strokes to produce perceptually salient visualizations. Psychophysical experiments confirm a strong relationship between the expressive power of our nonphotorealistic properties and previous findings on the use of perceptual color and texture patterns for data display. Results from these studies are used to produce effective nonphotorealistic visualizations. We conclude by applying our techniques to a large, multidimensional weather dataset to demonstrate their viability in a practical, real-world setting.
Puzzles in software development contracting
Authors: Lichtenstein Yossi
Published: February 2004

Current outsourcing practices are relatively unsophisticated in comparison with the techniques prescribed by economic theory. Customers end up bearing all long-term risk, and vendors have no direct incentive to achieve long-term system effectiveness.
A look at the economics of open source
Authors: Glass Robert L.
Published: February 2004

Is open source the future of the software field or a passing fad?
Coexpression of complementary fragments of ClC-5 and restoration of chloride channel function in a Dent's disease mutation
Authors: Mo L. , Xiong W. , Qian T. , Sun H. , Wills N. K.
Published: January 2004

The human hereditary disorder Dent's disease is linked to loss-of-function mutations of the chloride channel ClC-5. Many of these mutations involve insertion of premature stop codons, resulting in truncation of the protein. We determined whether the functional activity of ClC-5 could be restored by coexpression of the truncated protein (containing the NH2-terminal region) with its complementary “missing” COOH-terminal region. Split channel constructs for ClC-5, consisting of complementary N and C protein regions, were created at an arbitrary site in the COOH-terminal region (V655) and at four Dent's disease mutation sites (R347, Y617, R648, and R704). Coexpression of complementary fragments for the split channel at V655 produced currents with anion and pH sensitivity similar to those of wild-type ClC-5. Channel activity was similarly restored when complementary split channel constructs made for Dent's mutation R648 were coexpressed, but no ClC-5 currents were found when split channels for mutations R347, Y617, or R704 were coexpressed. Immunoblot and immunofluorescence studies of COS-7 cells revealed that N or C protein fragments could be transiently expressed and detected in the plasma membrane, even in split channels that failed to show functional activity. The results suggest that ClC-5 channel activity can be restored for specific Dent's mutations by expression of the missing portion of the ClC-5 molecule.
Discrete but simultaneous release of adenine nucleotides and serotonin from mouse megakaryocytes as detected with patch- and carbon-fiber electrodes
Authors: Kawa Kazuyoshi
Published: January 2004

Using patch- and carbon-fiber electrodes, we studied release phenomena of adenine nucleotides and serotonin from megakaryocytes isolated from the bone marrow of the mouse. Megakaryocytes express ionotropic purinergic receptors on their surfaces. Under the condition of whole cell recording, the cells showed spikelike spontaneous inward currents. The spontaneous currents were carried by cations and had amplitudes of 30–800 pA at –43 mV and durations of 0.1–0.3 s. Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 100 μM) and suramin (100 μM), purinoceptor-blocking agents, depressed the currents reversibly. It is thought that the receptor involved was the P2X1 subtype on the cell and that the currents were due to activation of the P2X1 receptor by adenine nucleotides released from the cell. The currents showed a skewed amplitude distribution, suggesting variation of vesicular contents and/or distinct localization or varied density of receptors on the cell. Frequency of the spontaneous inward currents was enhanced by external application of platelet-activating substances, thrombin (0.4 U/ml), phorbol ester (100 nM), and ADP (2 μM), at low concentrations. With a carbon-fiber electrode, which can detect oxidizable substances including serotonin, spikelike oxidation currents from the external surface of the megakaryocyte were detected. The frequency of the oxidation currents increased remarkably after the application of thrombin (10 U/ml). The majority of the oxidation currents coincided with the rising phase of the whole cell currents, suggesting corelease of serotonin and adenine nucleotide from the same vesicle. We concluded that megakaryocytes store adenine nucleotides and serotonin in the same vesicle and release them simultaneously in a discrete manner.
Dietary cholesterol alters Na+/K+ selectivity at intracellular Na+/K+ pump sites in cardiac myocytes
Authors: Buhagiar Kerrie A. , Hansen Peter S. , Kong Benjamin Y. , Clarke Ronald J. , Fernandes Clyne , Rasmussen Helge H.
Published: January 2004

A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na+/K+ pump to intracellular Na+, whereas a large increase in cholesterol levels decreases the sensitivity to Na+. To examine the mechanisms, we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped with the use of patch pipettes that contained osmotically balanced solutions with Na+ in a concentration of 10 mM and K+ in concentrations ([K+]pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na+/K+ current ( Ip) when pipette solutions were K+ free. A modest increase in serum cholesterol caused a [K+]pip-dependent increase in Ip, whereas a large increase caused a [K+]pip-dependent decrease in Ip. Modeling suggested that pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant KK describing the backward reaction E1 + 2K+ → E2(K+)2, whereas pump inhibition with a large increase in serum cholesterol can be explained by an increase in KK. Because hypercholesterolemia upregulates angiotensin II receptors and because angiotensin II regulates the Na+/K+ pump in cardiac myocytes in a [K+]pip-dependent manner, we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Because the ϵ-isoform of protein kinase C (ϵPKC) mediates effects of angiotensin II on the pump, we included specific ϵPKC-blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition.
Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane
Authors: Opazo Saez Anabelle , Zhang Wenwu , Wu Yidi , Turner Christopher E. , Tang Dale D. , Gunst Susan J.
Published: January 2004

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca2+ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction.
Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin
Authors: Hagi-Pavli Eleni , Farthing Paula M. , Kapas Supriya
Published: January 2004

Adrenomedullin (AM) and corticotrophin (ACTH) are both vasoactive peptides produced by a variety of cell types, including endothelial cells. Although AM and ACTH are considered to be important in the control of blood pressure and the response to stress, respectively, their role in inflammation and the immune response has not been clarified. This study shows, with the use of a cell-based ELISA, that AM and ACTH induce cell surface expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVEC). Furthermore, this effect appears to be mediated in part via elevation of cAMP, given that both peptides elevate cAMP, the cell-permeable cAMP analog dibutyryl cAMP is able to mimic induction of all three cell adhesion molecules and the effect of AM and ACTH is inhibited by the adenylyl cyclase inhibitor SQ-22536. These findings demonstrate a role for AM and ACTH in the regulation of the immune and inflammatory response.
p38 MAP kinase-mediated negative inotropic effect of HIV gp120 on cardiac myocytes
Authors: Kan Hong , Xie Zirong , Finkel Mitchell S.
Published: January 2004

Myocardial dysfunction leading to dilated cardiomyopathy has been documented with surprisingly high frequency in human immunodeficiency virus (HIV)-infected individuals. p38 MAP kinase has been implicated as a mediator of myocardial dysfunction. We previously reported p38 MAP kinase activation by the HIV coat protein gp120 in neonatal rat cardiac myocytes. We now report the direct inotropic effects of HIV gp120 on adult rat ventricular myocytes (ARVM). ARVM were continuously superfused with gp120, and percent fractional shortening (FS) was determined by automated border detection and simultaneous intracellular ionized free Ca2+ concentration ([Ca2+]i) measured by fura 2-AM fluorescence: gp120 alone increased FS and increased [Ca2+]i within 5 min and then depressed FS without a decrease in [Ca2+]i by 20–60 min, which persisted for at least 2 h. Exposure of ARVM to gp120 also resulted in the phosphorylation of the upstream regulator of p38 MAP kinase MKK3/6, p38 MAP kinase itself, and its downstream effector, ATF-2, over a similar time course. ERK (p44/42) and JNK stress signaling pathways were not similarly activated. The effects of the p38 MAP kinase inhibitor were concentration dependent. SB-203580 (10 μM) blocked both p38 MAP kinase phosphorylation and the delayed negative inotropic effect of gp120. SB-203580 (5 μM) selectively blocked phosphorylation of ATF-2 without blocking the phosphorylation of MKK3/6 or p38 MAP kinase itself. SB-203580 (5 μM) administered before, with, or after gp120 blocked the negative inotropic effect of gp120 in ARVM. p38 MAP kinase activation may be a common stress-response mechanism contributing to myocardial dysfunction in HIV and other nonischemic as well as ischemic cardiomyopathies.
Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase
Authors: Reinhardt Timothy A. , Horst Ronald L. , Waters W. Ray
Published: January 2004

On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca2+-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker α-mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca2+-ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La3+ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells.
Sphingomyelinase activates GLUT4 translocation via a cholesterol-dependent mechanism
Authors: Liu Ping , Leffler Brian J. , Weeks Lara K. , Chen Guoli , Bouchard Christine M. , Strawbridge Andrew B. , Elmendorf Jeffrey S.
Published: January 2004

A basis for the insulin mimetic effect of sphingomyelinase on glucose transporter isoform GLUT4 translocation remains unclear. Because sphingomyelin serves as a major determinant of plasma membrane cholesterol and a relationship between plasma membrane cholesterol and GLUT4 levels has recently become apparent, we assessed whether GLUT4 translocation induced by sphingomyelinase resulted from changes in membrane cholesterol content. Exposure of 3T3-L1 adipocytes to sphingomyelinase resulted in a time-dependent loss of sphingomyelin from the plasma membrane and a concomitant time-dependent accumulation of plasma membrane GLUT4. Degradation products of sphingomyelin did not mimic this stimulatory action. Plasma membrane cholesterol amount was diminished in cells exposed to sphingomyelinase. Restoration of membrane cholesterol blocked the stimulatory effect of sphingomyelinase. Increasing concentrations of methyl-β-cyclodextrin, which resulted in a dose-dependent reversible decrease in membrane cholesterol, led to a dose-dependent reversible increase in GLUT4 incorporation into the plasma membrane. Although increased plasma membrane GLUT4 content by cholesterol extraction with concentrations of methyl-β-cyclodextrin above 5 mM most likely reflected decreased GLUT4 endocytosis, translocation stimulated by sphingomyelinase or concentrations of methyl-β-cyclodextrin below 2.5 mM occurred without any visible changes in the endocytic retrieval of GLUT4. Furthermore, moderate loss of cholesterol induced by sphingomyelinase or low concentrations of methyl-β-cyclodextrin did not alter membrane integrity or increase the abundance of other plasma membrane proteins such as the GLUT1 glucose transporter or the transferrin receptor. Regulation of GLUT4 translocation by moderate cholesterol loss did not involve known insulin-signaling proteins. These data reveal that sphingomyelinase enhances GLUT4 exocytosis via a novel cholesterol-dependent mechanism.
Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure
Authors: Dalla Libera Luciano , Ravara Barbara , Volterrani Maurizio , Gobbo Valerio , Della Barbera Mila , Angelini Annalisa , Betto Daniela Danieli , Germinario Elena , Vescovo Giorgio
Published: January 2004

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor-α (TNF-α) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg·kg–1·day–1 and 1.0 mg·kg–1·day–1) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF-α and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF-α and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity.
Actin reorganization and morphological changes in human neutrophils stimulated by TNF, GM-CSF, and G-CSF: the role of MAP kinases
Authors: Kutsuna Haruo , Suzuki Kenichi , Kamata Noriko , Kato Takayuki , Hato Fumihiko , Mizuno Kensaku , Kobayashi Hiromi , Ishii Masamitsu , Kitagawa Seiichi
Published: January 2004

Stimulation of human neutrophils with tumor necrosis factor-α (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) resulted in decreased fluorescence intensity of FITC-phalloidin (actin depolymerization) and morphological changes. Cytokine-induced actin depolymerization was dependent on the concentration of cytokines used as stimuli. The maximal changes were detected at 10 min after stimulation with TNF or GM-CSF and at 20 min after stimulation with G-CSF. Cytokine-induced actin depolymerization was sustained for at least 30 min after stimulation. In contrast, N-formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly (within 45 s) induced an increase in the fluorescence intensity of FITC-phalloidin (actin polymerization) and morphological changes. TNF- and GM-CSF-induced actin depolymerization and morphological changes, but not FMLP-induced responses, were partially inhibited by either PD-98059, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase, or SB-203580, an inhibitor of p38 MAPK, and were almost completely abolished by these inhibitors in combination. G-CSF-induced responses were almost completely abolished by PD-98059 and were unaffected by SB-203580. These findings are consistent with the ability of these cytokines to activate the distinct MAPK subtype cascade in human neutrophils. Phosphorylated ERK and p38 MAPK were not colocalized with F-actin in neutrophils stimulated by cytokines or FMLP. Furthermore, FMLP-induced polarization and actin polymerization were prevented by cytokine pretreatment. These findings suggest that TNF, GM-CSF, and G-CSF induce actin depolymerization and morphological changes through activation of ERK and/or p38 MAPK and that cytokine-induced actin reorganization may be partly responsible for the inhibitory effect of these cytokines on neutrophil chemotaxis.
PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration
Authors: Qian Yong , Corum Linda , Meng Qiao , Blenis John , Zheng Jenny Z. , Shi Xianglin , Flynn Daniel C. , Jiang Bing-Hua
Published: January 2004

This study was designed to identify the molecular mechanisms of phosphatidylinositol 3-kinase (PI3K)-induced actin filament remodeling and cell migration. Expression of active forms of PI3K, v-P3k or Myr-P3k, was sufficient to induce actin filament remodeling to lead to an increase in cell migration, as well as the activation of Akt in chicken embryo fibroblast (CEF) cells. Either the inhibition of PI3K activity using a PI3K-specific inhibitor, LY-294002, or the disruption of Akt activity restored the integrity of actin filaments in CEF cells and inhibited PI3K-induced cell migration. We also found that expression of an activated form of Akt (Myr-Akt) was sufficient to remodel actin filaments to lead to an increase in cell migration, which was unable to be inhibited by the presence of LY-294002. Furthermore, we found that p70S6K1 kinase was a downstream molecule that can mediate the effects of both PI3K and Akt on actin filaments and cell migration. Overexpression of an active form of p70S6K1 was sufficient to induce actin filament remodeling and cell migration in CEF cells, which requires Rac activity. These results demonstrate that activation of PI3K activity alone is sufficient to remodel actin filaments to increase cell migration through the activation of Akt and p70S6K1 in CEF cells.
Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells
Authors: Dedkova Elena N. , Ji Xiang , Lipsius Stephen L. , Blatter Lothar A.
Published: January 2004

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]mt). Stimulation of mitochondrial Ca2+ uptake by exposure to different cytoplasmic Ca2+ concentrations (1, 2, and 5 μM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]mt was sensitive to the NOS antagonist l- N5-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca2+ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl-β-cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca2+ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis.
Differential Ca2+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells
Authors: Kim Yuri V. , Di Cello Francescopaolo , Hillaire Coryse S. , Kim Kwang Sik
Published: January 2004

Thrombin and related protease-activated receptors 1, 2, 3, and 4 (PAR1–4) play a multifunctional role in many types of cells including endothelial cells. Here, using RT-PCR and immunofluorescence staining, we showed for the first time that PAR1–4 are expressed on primary human brain microvascular endothelial cells (HBMEC). Digital fluorescence microscopy and fura 2 were used to monitor intracellular Ca2+ concentration ([Ca2+]i) changes in response to thrombin and PAR1-activating peptide (PAR1-AP) SFFLRN. Both thrombin and PAR1-AP induced a dose-dependent [Ca2+]i rise that was inhibited by pretreatment of HBMEC with the phospholipase C inhibitor U-73122 and the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thrombin induced transient [Ca2+]i increase, whereas PAR1-AP exhibited sustained [Ca2+]i rise. The PAR1-AP-induced sustained [Ca2+]i rise was significantly reduced in the absence of extracellular calcium or in the presence of an inhibitor of store-operated calcium channels, SKF-96365. Restoration of extracellular Ca2+ to the cells that were initially activated by PAR1-AP in the absence of extracellular Ca2+ resulted in significant [Ca2+]i rise; however, this effect was not observed after thrombin stimulation. Pretreatment of the cells with a low thrombin concentration (0.1 nM) prevented [Ca2+]i rise in response to high thrombin concentration (10 nM), but pretreatment with PAR1-AP did not prevent subsequent [Ca2+]i rise to high PAR1-AP concentration. Additionally, treatment with thrombin decreased transendothelial electrical resistance in HBMEC, whereas PAR1-AP was without significant effect. These findings suggest that, in contrast to thrombin, stimulation of PAR1 by untethered peptide SFFLRN results in stimulation of store-operated Ca2+ influx without significantly affecting brain endothelial barrier functions.
Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts
Authors: Short Megan , Nemenoff Raphel A. , Zawada W. Michael , Stenmark Kurt R. , Das Mita
Published: January 2004

Activation of the α-smooth muscle actin (α-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates α-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased α-SMA expression is controlled at the transcriptional level because the α-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the α-SMA gene was mimicked by overexpression of constitutively active Gαi2 (αi2Q205L) but not Gα16 (α-16Q212L). Blockade of hypoxia-induced α-SMA expression with pertussis toxin, a Gαi antagonist, confirmed a role for Gαi in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated α-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and α-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-β1 blocked only 30% of the hypoxia-induced α-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of α-SMA, and this increase in α-SMA level occurs through Gαi- and JNK-dependent signaling pathways.
The influence of Lyn kinase on Na,K-ATPase in porcine lens epithelium
Authors: Bozulic Larry D. , Dean William L. , Delamere Nicholas A.
Published: January 2004

Na,K-ATPase is essential for the regulation of cytoplasmic Na+ and K+ levels in lens cells. Studies on the intact lens suggest activation of tyrosine kinases may inhibit Na,K-ATPase function. Here, we tested the influence of Lyn kinase, a Src-family member, on tyrosine phosphorylation and Na,K-ATPase activity in membrane material isolated from porcine lens epithelium. Western blot studies indicated the expression of Lyn in lens cells. When membrane material was incubated in ATP-containing solution containing partially purified Lyn kinase, Na,K-ATPase activity was reduced by ∼38%. Lyn caused tyrosine phosphorylation of multiple protein bands. Immunoprecipitation and Western blot analysis showed Lyn treatment causes an increase in density of a 100-kDa phosphotyrosine band immunopositive for Na,K-ATPase α1 polypeptide. Incubation with protein tyrosine phosphatase 1B (PTP-1B) reversed the Lyn-dependent tyrosine phosphorylation increase and the change of Na,K-ATPase activity. The results suggest that Lyn kinase treatment of a lens epithelium membrane preparation is able to bring about partial inhibition of Na,K-ATPase activity associated with tyrosine phosphorylation of multiple membrane proteins, including the Na,K-ATPase α1 catalytic subunit.
Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module
Authors: Lee Eun Hui , Lopez J. Rafael , Li Jingzi , Protasi Feliciano , Pessah Isaac N. , Kim Do Han , Allen P. D.
Published: January 2004

Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca2+ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La3+ and Cd2+ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La3+ and Cd2+ or nifedipine in the external medium at concentrations known to block Ca2+ entry through the DHPRs significantly decreased the caffeine EC50 of wtRyR1 (2.80 ± 0.12 to 0.83 ± 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC50 values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC50 values of chimeras R4 and R9 were significantly increased (1.27 ± 0.05 to 2.60 ± 0.16 mM, and 1.15 ± 0.03 to 2.11 ± 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca2+ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [3H]ryanodine regardless of Ca2+ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca2+ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism.
Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury
Authors: Hu Zhihong , Sayeed Mohammed M.
Published: January 2004

Neutrophil apoptosis is delayed under trauma and/or sepsis conditions. The mechanism for the delay has remained unclear. We hypothesize that modulation of the mitochondrial pathway of apoptosis contributes to the delay in neutrophil apoptosis with burn injury. Rats were subjected to burn injury (30% of total body surface area, 98°C for 10 s) and euthanatized 24 h postinjury. Blood neutrophils from sham and burn-injured rats were isolated by Ficoll gradient centrifugation and cultured for 2 or 8 h. Neutrophil apoptosis was determined by annexin V and propidium iodide (PI) labeling and flow cytometry. Neutrophil mitochondrial morphology was assessed via histochemical staining (MitoTracker GreenFM) and confocal microscopy. Neutrophils from rats with burn injury showed a decreased level of apoptosis compared with sham rat neutrophils at both 2 and 8 h of incubation. In incubated sham rat neutrophils, mitochondria showed a change from normal “tubular” to an “aggregated” morphology. In contrast, cultured neutrophils from burn rats did not exhibit this mitochondrial morphological transition until 8 h of incubation. Compared with sham rat neutrophils, neutrophils from burn rats showed decreased levels of active caspase-9 and -3. Whereas an upregulation of Bcl-xL and a downregulation of Bax seemed to contribute to decreased apoptosis in burn rat neutrophils at 2 h of incubation, the decreased apoptosis at 8 h appeared to be associated with a decrease in Bax and increased phosphorylated Bad. These data suggest that suppression of the mitochondrial pathway plays an essential role in the delay of polymorphonuclear neutrophil apoptosis with burn injury.