bluuebunny/crossref_metadata_2025 huggingface dataset:
ALY Is a Common Coactivator of RUNX1 and c-Myb on the Type B Leukemogenic VirusEnhancer
Authors: Mertz Jennifer A. , Kobayashi Ryuji , Dudley Jaquelin P.
Published: January 2007

Type B leukemogenic virus (TBLV), a mouse mammary tumor virus (MMTV) variant, often induces T-cell leukemias and lymphomas by c-mycactivation following viral DNA integration. Transfection assays using a c-mycreporter plasmid indicated that the TBLV long terminal repeat (LTR) enhancer is necessary for T-cell-specific increases in basal reporter activity. The sequence requirements for this effect were studied using mutations of the 62-bp enhancer region in an MMTV LTR reporter vector. Deletion of a nuclear factor A-binding site dramatically reduced reporter activity in Jurkat T cells. However, a 41-bp enhancer missing the RUNX1 site still retained minimal enhancer function. DNA affinity purification using a TBLV enhancer oligomer containing the RUNX1 binding site followed by mass spectrometry resulted in the identification of ALY. Subsequent experiments focused on the reconstitution of enhancer activity in epithelial cells. ALY overexpression synergized with RUNX1B on TBLV enhancer activity, and synergism required the RUNX1B-binding site. A predicted c-Myb binding site in the enhancer was confirmed after c-myboverexpression elevated TBLV LTR reporter activity, and overexpression of c-Myb and RUNX1B together showed additive effects on reporter gene levels. ALY also synergized with c-Myb, and coimmunoprecipitation experiments demonstrated an interaction between ALY and c-Myb. These experiments suggest a central role for ALY in T-cell enhancer function and oncogene activation.
Mutations That Decrease DNA Binding of the Processivity Factor of the Herpes Simplex Virus DNA Polymerase Reduce Viral Yield, Alter the Kinetics of Viral DNA Replication, and Decrease the Fidelity of DNA Replication
Authors: Jiang Changying , Hwang Ying T. , Randell John C. W. , Coen Donald M. , Hwang Charles B. C.
Published: January 2007

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a
UL42
null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a
lacZ
insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type
UL42
. Real-time PCR assays revealed that these
UL42
mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne
lacZ
gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.
The Mimivirus Genome Encodes a Mitochondrial Carrier That Transports dATP and dTTP
Authors: Monné Magnus , Robinson Alan J. , Boes Christoph , Harbour Michael E. , Fearnley Ian M. , Kunji Edmund R. S.
Published: January 2007

Members of the mitochondrial carrier family have been reported in eukaryotes only, where they transport metabolites and cofactors across the mitochondrial inner membrane to link the metabolic pathways of the cytosol and the matrix. The genome of the giant virus
Mimiviridae mimivirus
encodes a member of the mitochondrial carrier family of transport proteins. This viral protein has been expressed in
Lactococcus lactis
and is shown to transport dATP and dTTP. As the 1.2-Mb double-stranded DNA mimivirus genome is rich in A and T residues, we speculate that the virus is using this protein to target the host mitochondria as a source of deoxynucleotides for its replication.
Targeting of the Sendai Virus C Protein to the Plasma Membrane via a Peptide-Only Membrane Anchor
Authors: Marq Jean-Baptiste , Brini Albert , Kolakofsky Daniel , Garcin Dominique
Published: January 2007

Several cellular proteins are synthesized in the cytosol on free ribosomes and then associate with membranes due to the presence of short peptide sequences. These membrane-targeting sequences contain sites to which lipid chains are attached, which help direct the protein to a particular membrane domain and anchor it firmly in the bilayer. The intracellular concentration of these proteins in particular cellular compartments, where their interacting partners are also concentrated, is essential to their function. This paper reports that the apparently unmodified N-terminal sequence of the Sendai virus C protein (MPS
FL

KK

IL

K

L

R

G

RR

. . .; letters in italics represent hydrophobic residues; underlined letters represent basic residues, which has a strong propensity to form an amphipathic α-helix in a hydrophobic environment) also function as a membrane targeting signal and membrane anchor. Moreover, the intracellular localization of the C protein at the plasma membrane is essential for inducing the interferon-independent phosphorylation of Stat1 as part of the viral program to prevent the cellular antiviral response.
Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Critical for Ebola Virus Replication in Nonhuman Primates
Authors: Neumann Gabriele , Geisbert Thomas W. , Ebihara Hideki , Geisbert Joan B. , Daddario-DiCaprio Kathleen M. , Feldmann Heinz , Kawaoka Yoshihiro
Published: January 2007

Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequence compared to those infected with wild-type virus. We conclude that glycoprotein cleavage by subtilisin-like endoproteases is not critical for Ebola virus infectivity and virulence in nonhuman primates.
Inhibition of Cellular 2′-5′ Oligoadenylate Synthetase by the Herpes Simplex Virus Type 1 Us11 Protein
Authors: Sànchez Ricardo , Mohr Ian
Published: January 2007

Among the many host genes induced by virus infection and interferon, the eIF2α protein kinase PKR and the 2′-5′ oligoadenylate synthetase (OAS) are both activated by double-stranded RNA (dsRNA) produced in virus-infected cells. Furthermore, each is a critical component that independently acts to inhibit virus replication and thereby contributes to the establishment of an antiviral state. As part of their tactics to foil host defense mechanisms, some viruses prevent the induction of interferon-responsive genes at the level of transcription. Other viruses, such as herpes simplex virus type 1 (HSV-1), can additionally replicate in interferon-treated cells and must also evade the actions of host defense proteins such as PKR and OAS that have been previously synthesized and merely await detection of an activating signal. Whereas HSV-1 gene products γ
1
34.5 and Us11 are required for viral replication in interferon-treated cells and both act in a temporally coordinated manner during infection to counteract PKR, HSV-1 functions that target OAS have not been described. Here, we demonstrate that HSV-1 infection inhibits 2′-5′ oligoadenylate synthesis in interferon-stimulated primary human cells. The OAS-inhibiting activity is generated late in the virus' productive life cycle and requires the Us11 gene product. Moreover, we establish that the Us11 protein is sufficient to block OAS activation in extracts from uninfected, interferon-treated cells. Inhibition of OAS specifically requires the Us11 dsRNA-binding domain, suggesting a mechanism that, in part, relies on sequestering available dsRNA produced during infection. Thus, in addition to PKR and its protein activator, PACT, the HSV-1 Us11 gene product is able to counteract the activity of OAS, a third cellular protein critical for host defense.
Immediate Activation Fails To Rescue Efficient Human Immunodeficiency Virus Replication in Quiescent CD4+T Cells
Authors: Vatakis Dimitrios N. , Bristol Gregory , Wilkinson Thomas A. , Chow Samson A. , Zack Jerome A.
Published: January 2007

Unlike activated T cells, quiescent CD4+T cells have shown resistance to human immunodeficiency virus (HIV) infection due to a block in the early events of the viral life cycle. To further investigate the nature of this block, we infected quiescent CD4+T cells with HIV-1NL4-3and immediately stimulated them. Compared to activated (prestimulated) cells, these poststimulated cells showed slightly decreased viral entry and delays in the completion of reverse transcription. However, the relative efficiency of integration was similar to that of prestimulated cells. Together, this resulted in decreased expression oftat/revmRNA and synthesis of viral protein. Furthermore, based on cell cycle staining and BrdU incorporation, poststimulated cells expressing viral protein failed to initiate a second round of their cell cycle, independently of Vpr-mediated arrest. Together, these data demonstrate that the early stages of the HIV life cycle are inefficient in these poststimulated cells and that efficient replication cannot be induced by subsequent activation.
A Second Receptor Binding Site on Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Contributes to Activation of the FusionMechanism
Authors: Porotto Matteo , Fornabaio Micaela , Kellogg Glen E. , Moscona Anne
Published: January 2007

The
hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out
three discrete activities that each affect the ability of HN to promote
viral fusion and entry: receptor binding, receptor cleaving
(neuraminidase), and triggering of the fusion protein. The
interrelationship between the receptor binding and fusion-triggering
functions of HN has not been clear. For human parainfluenza type 3
(HPIV3), one bifunctional site on HN can carry out both receptor
binding and neuraminidase activities, and this site's receptor binding
can be inhibited by the small receptor analog zanamivir. We now report
experimental evidence, complemented by computational data, for a second
receptor binding site near the HPIV3 HN dimer interface. This second
binding site can mediate receptor binding even in the presence of
zanamivir, and it differs from the second receptor binding site of the
paramyxovirus Newcastle disease virus in its function and its
relationship to the primary binding site. This second binding site of
HPIV3 HN is involved in triggering F. We suggest that the two
receptor binding sites on HPIV3 HN each contribute in distinct ways to
virus-cell interaction; one is the multifunctional site that contains
both binding and neuraminidase activities, and the other contains
binding activity and also is involved in fusion
promotion.
FIRST PRINCIPLES CALCULATIONS OF ADSORPTION AND DIFFUSION OF Ba ON A RECONSTRUCTED Si(001) SURFACE
Authors: WANG Z. G. , ZU X. T.
Published: January 2007

Adsorption and diffusion of a Ba adatom on a reconstructed Si (001) surface were studied using first principles density functional calculation. It was found that the lowest energetic configuration is that the Ba atom resides at the T3' (which is displaced along the [Formula: see text] direction from the T3 site by ~ 0.26 Å). An anisotropic diffusivity of Ba on the reconstructed surface was found. The preferred direction of diffusion was the trough between Si dimer rows.
GROWTH CONTROL OF ZnO FILMS ON GLASS BY CHEMICAL BATH DEPOSITION
Authors: YU MUNAN , XU HAIYAN , WANG HAO , YAN HUI , PEI ZHILIANG , GONG JUN , SUN CHAO
Published: January 2007

The effects of substrate-placing manner, deposition temperature and solution concentration on the preparation of ZnO films by chemical bath deposition were investigated in this work. The structures and the morphologies of as-deposited ZnO films were characterized by X-ray diffraction and scanning electron microscope, respectively. The results show that compared with vertically insertion, floating the substrates on the solutions could greatly improve the c-axis orientation of the films. The growth of ZnO films with orientation would also be favored at lower precursor concentrations and temperatures. When the concentration of Zn ( CH 3 COO )2·2 H 2 O is 0.033 M/L and the deposited temperature is 60°C, ZnO films which are well crystallized and highly c-axis orientated were obtained.
MICROSCOPIC MODEL OF ASSOCIATIVE DESORPTION FOR HYDROGEN ON Mo(110)
Authors: YAKOVKIN I. N. , OSOVSKII V. D. , PETROVA N. V. , PTUSHINSKII Yu. G.
Published: January 2007

Adsorbed hydrogen layers on the Mo (110) surface have been investigated both experimentally by temperature programmed desorption (TPD) method and theoretically by means of DFT-based optimization of surface structures. We suggest a novel microscopic model of the associative hydrogen desorption, which explains essential features of the process. In this model, the process of hydrogen desorption can be described as association of hydrogen atoms on the surface, but molecular formation is actually accomplished while the molecule moves away from the surface. We also suggest a new algorithm for realistic Monte Carlo simulations of associative desorption, which implements the microscopic description of the association of hydrogen adatoms into a molecule with activation energy, found from the DFT calculations. Good agreement between simulated and experimental TPD spectra gives insight into different behavior of the spectra, obtained for low and high hydrogen coverages on the Mo (110) surface.
SULFUR ADSORPTION ON THE GOETHITE (110) SURFACE
Authors: SIMONETTI S. , DAMIANI D. , BRIZUELA G. , JUAN A.
Published: January 2007

The electronic structure of S adsorption on goethite (110) surface has been studied by ASED-MO cluster calculations. For S location, the most exposed surface atoms of goethite surface were selected. The calculations show that the surface offers several places for S adsorption. The most energetically stable system corresponds to S location above H atom.

We studied in detail the configurations that correspond to the higher OP values. For these configurations, the H-S and Fe-S computed distances are 2.1 and 3.7 Å, respectively. The H-S and Fe-S are mainly bonding interaction with OP values of 0.156 and 0.034, respectively. The Fe-S interaction mainly involves Fe 3dx2-y2atomic orbitals with lesser participation of Fe 4pyand Fe 3dyzatomic orbitals. The O-S interaction shows the same bonding and antibonding contributions giving a small OP value. The O-S interaction involves O 2p orbitals. There is an electron transfer to the Fe atom from the S atom. On the other hand, there is an electron transfer to S atom from the H and O atoms, respectively.
THE EXPERIMENT OF PLASMA-SPRAYED HA COATINGS ON CARBON/CARBON COMPOSITES IN BONE
Authors: CAO NING , MA QUAN-SHENG , SUI JIN-LING , WANG QIANG-XIU , LÜ YU-PENG , CHEN YAN-MEI , LI MU-SEN
Published: January 2007

This paper dealt with plasma-sprayed hydroxyapatite (HA) coatings on carbon/carbon composites ( C/C ) and the implantation was carried out in rabbits. Ninety days after the implantation, we found that such materials were favorable for health and no cytotoxic effects were discovered. The histological observation of osteogenic response of HA-coated C/C showed that the osteoplaque had scansorially grown along the surface of the HA coatings and the pure C/C surface had no obvious change. All these results indicate that as a potential bone-repairing material, the surface properties of the C/C has been changed by preparing the HA coatings. Also the new materials may be preferably feasible of clinical application.
AC CONDUCTION IN MIXED OXIDES Al–In2O3–SnO2–Al STRUCTURE DEPOSITED BY CO-EVAPORATION
Authors: ANWAR M. , SIDDIQI S. A. , GHAURI I. M.
Published: January 2007

Conductivity-frequency and capacitance-frequency characteristics of mixed oxides Al – In 2 O 3– SnO 2– Al structure are examined to elicit any correlation with the conduction mechanisms most often observed in thin film work. The existence of Schottky barriers is believed to be due to a strong donor band in the insulator established during the vacuum evaporation when a layer of mixed oxides In 2 O 3– SnO 2 system is sandwiched between two metal electrodes. Low values of activation energy at low temperatures indicate that the transport of the carriers between localized states is mainly due to electronic hopping over the barrier separating the two nearest neighbor sites. The increase in the formation of ionized donors with increase in temperature during electrical measurements indicates that electronic part of the conductivity is higher than the ionic part. The initial increase in conductivity with increase in Sn content in In 2 O 3 lattice is caused by the Sn atom substitution of In atom, giving out one extra electron. The decrease in electrical conductivity above the critical Sn content (10 mol% SnO 2) is caused by the defects formed by Sn atoms, which act as carrier traps rather than electron donors. The increase in electrical conductivity with film thickness is caused by the increase in free carriers density, which is generated by oxygen vacancy acting as two electron donor. The increase in conductivity with substrate and annealing temperatures is due to either the severe deficiency of oxygen, which deteriorates the film properties and reduces the mobility of the carriers or to the diffusion of Sn atoms from interstitial locations into the In cation sites and formation of indium species of lower oxidation state ( In 2+). Calculations of C and σac from tan δ measurements suggest that there is some kind of space-charge polarization in the material, caused by the storage of carriers at the electrodes. Capacitance decreases not only with the rise of frequency but also with the lowering of temperature. At low temperatures the major contribution to capacitance arises from the ionic polarization, however, with the increase of temperature the contribution from orientation polarization would considerably increase. The decrease in capacitance with the increase in frequency may be attributed to interfacial polarization.
AN INVESTIGATION OF SMOOTH NANOSIZED COPPER FILMS ON GLASS SUBSTRATE BY IMPROVED ELECTROLESS PLATING
Authors: ZHANG HUIPING , JIANG ZHONGHAO , LIU XIANLI , LIAN JIANSHE
Published: January 2007

Thin nanocrystalline Cu films (< 1 μm) are deposited on a glass substrate using an improved electroless plating technique. The deposition course of the Cu film is illustrated by the variation of surface morphology with different deposition time. The results show that a more uniform and continuous nanocrystalline Cu film with very small nodules can be formed on a glass substrate at the deposition time over 1 min. The roles of SDBS as an additive in the bath are also discussed. According to the relation of the film thickness and the deposition time, it is obvious that the film thickness nearly linearly varies with the deposition time in the present work. An enhanced (111) texture with the diffraction intensity ratio (I(111)/I(200)) of about 4.0 and the very fine grain size of 15–28 nm determined by X-ray results has been observed. The variations of the resistivity show that it is strongly affected by the film thickness and grain size.
CHARACTERIZATION OF SURFACE PROPERTIES AND MICROSTRUCTURE OF PVD-TiN FILMS USING MEVVA ION IMPLANTATION
Authors: YANG J. H. , CHENG M. F. , LUO X. D. , ZHANG T. H.
Published: January 2007

The PVD- TiN film was implanted with titanium ions and the improvement in surface wear resistance was investigated. Ti ion implantation was done using a metal vapor vacuum arc (MEVVA) ion source with an implantation dose of 2 × 1016 ions/cm2 and at an extraction voltage of 48 kV. The wear characteristics of the implanted zone was measured and compared to the performance of the unimplanted zone by a pin-on-disc apparatus and an optical interference microscope. The structure of the implanted zone and unimplanted one was observed by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). A dynamic TRIM called TRIDYN was used to calculate the concentration depth profile of implanted Ti in TiN to investigate the profile of multi-charge state ions. The results showed that the improved wear resistance of the TiN film was mainly due to the presence of nano-order TiN crystal grains after Ti ion implantation.
EFFECT OF Ni ON THE ELECTRICAL AND MICROSTRUCTURAL PROPERTIES OF NANOCRYSTALLITES Fe2O3/TiO2 SYSTEM
Authors: SOBHY MAGED S.
Published: January 2007

Nominal compositions of Ni x Ti 1-x Fe 2 O 5-δ (x = 0, 0.2, 0.4, 0.6, 0.8 and 1) were prepared by a solid state reaction using stoichiometric amounts of Fe 2 O 3/ TiO 2 system and NiO as a dopant. The effects of small substitution of Ni ions on the electrical and structural properties were studied for the above system. The X-ray diffraction patterns revealed that the ferroelectric phase of iron titanate and the spinel ferrite phase of Ni -ferrite having a single phase at x = 0 and 1, respectively. The substitution of Ni ions increases the average value of lattice constant aav. Solid–solid interaction took place between the ternary oxides at 1200°C for 4 h yielding a new phase of NiTiO 3. The presence of the three phases was confirmed by X-ray diffraction technique. The resultant compositions have nanocrystallites with average crystalline size "D av " in the range 100–300 nm. The DC electrical resistivity ρ, Curie temperature TC and activation energies for electric conduction around TC region increase as Ni ion substitution increases. The ferrite samples have a semiconductor behavior where electrical resistivity ρ decreases on increasing temperature. The activation energy for electrical conduction was affected by both the ratio "ferroelectric/ferrite" and the position of the Curie temperatures in the compositions depending on the ( Ni , Ti ) to Fe ratio.
CORROSION BEHAVIOR OF Ni–W/SiC NANOCOMPOSITE COATING IN NaCl SOLUTION
Authors: YAO YINGWU , YAO SUWEI , ZHANG LU
Published: January 2007

Ni–W/SiC nanocomposite coatings were prepared by electrodeposition in Ni–W plating bath containing SiC nanoparticulates. The effect of incorporation of SiC particulates into the Ni–W matrix on the morphology of the coating surface and corrosion properties was investigated. Scanning electron microscopy (SEM) showed that the SiC nanoparticulates are uniformly distributed in the Ni–W matrix. Corrosion tests were performed in 0.5 M NaCl solution using electrochemical measurements for Ni–W alloy coating and Ni–W/SiC nanocomposite coating. The results showed that the co-deposition of SiC nanoparticulates in the composite coatings increased the corrosion resistance. At last, the anti-corrosion mechanism of the nanocomposite coatings is also discussed.
STRUCTURE AND ELEMENT DISTRIBUTION OF Al2O3 COATING ON ZL109 ALLOY BY PLASMA ELECTROLYSIS OXIDATION
Authors: DONG QING , CHEN CHUANZHONG , WANG DIANGANG , WANG XUBO
Published: January 2007

It is thought that the PEO (plasma electrolysis oxidation) coatings are difficult to be prepared on the cast Al – Si alloys because these alloys contain lots of pure silicon phases. In the present study, oxide coatings with a thickness of about 10 μm were deposited on the substrate of the ZL109 Al – Si alloy by a PEO treatment. XRD showed that the phase composition of the coating was γ- Al 2 O 3. SEM showed that the coating surface was porous, and the pore diameter was in the range of 1–5 μm. The cross-section view by EDS indicated that the coating matrix is quite dense, and there are few defects. Phosphorus from the applied electrolyte could be incorporated into the coating during the PEO process. A transitional zone with a thickness of about 3 μm was observed on the interface between the coating and substrate, and a high adhesive strength was expected. The average hardness of the coating was 576 HV0.025, which was 7.2 times higher than that of the substrate. High alloy element content of the substrate seems to have no remarkable and adverse influence on the structure and composition of the PEO coating.
THE EFFECT OF RARE EARTH ON THE STRUCTURE AND PERFORMANCE OF LASER CLAD COATINGS
Authors: BAO RUILIANG , YU HUIJUN , CHEN CHUANZHONG , DONG QING
Published: January 2007

Laser cladding is one kind of advanced surface modification technology and has the abroad prospect in making the wear-resistant coating on metal substrates. However, the application of laser cladding technology does not achieve the people's expectation in the practical production because of many defects such as cracks, pores and so on. The addiction of rare earth can effectively reduce the number of cracks in the clad coating and enhance the coating wear-resistance. In the paper, the effects of rare earth on metallurgical quality, microstructure, phase structure and wear-resistance are analyzed in turns. The preliminary discussion is also carried out on the effect mechanism of rare earth. At last, the development tendency of rare earth in the laser cladding has been briefly elaborated.
Obligatory role for phospholipase C-γ1 in villin-induced epithelial cell migration
Authors: Wang Yaohong , Tomar Alok , George Sudeep P. , Khurana Seema
Published: January 2007

While there is circumstantial evidence to suggest a requirement for phospholipase C-γ1 (PLC-γ1) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-γ1 play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-γ1. Inhibition of villin phosphorylation prevented villin-PLC-γ1 complex formation as well as villin-induced cell migration. The absolute requirement for PLC-γ1 in villin-induced cell migration was demonstrated by measuring cell motility in PLC-γ1−/− cells and by downregulation of endogenous PLC-γ1. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-γ1 at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration.
Developmental regulation of the mouse IGF-I exon 1 promoter region by calcineurin activation of NFAT in skeletal muscle
Authors: Alfieri Christina M. , Evans-Anderson Heather J. , Yutzey Katherine E.
Published: January 2007

Skeletal muscle development and growth are regulated through multiple signaling pathways that include insulin-like growth factor I (IGF-I) and calcineurin activation of nuclear factor of activated T cell (NFAT) transcription factors. The developmental regulation and molecular mechanisms that control IGF-I gene expression in murine embryos and in differentiating C2C12 skeletal myocytes were examined. IGF-I is expressed in developing skeletal muscle, and its embryonic expression is significantly reduced in embryos lacking both NFATc3 and NFATc4. During development, the IGF-I exon 1 promoter is active in multiple organ systems, including skeletal muscle, whereas the alternative exon 2 promoter is expressed predominantly in the liver. The IGF-I exon 1 promoter flanking sequence includes two highly conserved regions that contain NFAT consensus binding sequences. One of these conserved regions contains a calcineurin/NFAT-responsive regulatory region that is preferentially activated by NFATc3 in C2C12 skeletal muscle cells and NIH3T3 fibroblasts. This NFAT-responsive region contains three clustered NFAT consensus binding sequences, and mutagenesis experiments demonstrated the requirement for two of these in calcineurin or NFATc3 responsiveness. Chromatin immunoprecipitation analyses demonstrated that endogenous IGF-I genomic sequences containing these conserved NFAT binding sequences interact preferentially with NFATc3 in C2C12 cells. Together, these experiments demonstrated that a NFAT-rich regulatory element in the IGF-I exon 1 promoter flanking region is responsive to calcineurin signaling and NFAT activation in skeletal muscle cells. The identification of a calcineurin/NFAT-responsive element in the IGF-I gene represents a potential mechanism of intersection of these signaling pathways in the control of muscle development and homeostasis.
Functional analysis of the aquaporin gene family in Caenorhabditis elegans
Authors: Huang Chunyi George , Lamitina Todd , Agre Peter , Strange Kevin
Published: January 2007

Aquaporin channels facilitate the transport of water, glycerol, and other small solutes across cell membranes. The physiological roles of many aquaporins remain unclear. To better understand aquaporin function, we characterized the aquaporin gene family in the nematode Caenorhabditis elegans. Eight canonical aquaporin-encoding genes ( aqp) are present in the worm genome. Expression of aqp-2, aqp-3, aqp-4, aqp-6, or aqp-7 in Xenopus oocytes increased water permeability five- to sevenfold. Glycerol permeability was increased three to sevenfold by expression of aqp-1, aqp-3, or aqp-7. Green fluorescent protein transcriptional and translational reporters demonstrated that aqp genes are expressed in numerous C. elegans cell types, including the intestine, excretory cell, and hypodermis, which play important roles in whole animal osmoregulation. To define the role of C. elegans aquaporins in osmotic homeostasis, we isolated deletion alleles for four aqp genes, aqp-2, aqp-3, aqp-4, and aqp-8, which are expressed in osmoregulatory tissues and mediate water transport. Single, double, triple, and quadruple aqp mutant animals exhibited normal survival, development, growth, fertility, and movement under normal and hypertonic culture conditions. aqp-2; aqp-3; aqp-4; aqp-8 quadruple mutants exhibited a slight defect in recovery from hypotonic stress but survived hypotonic stress as well as wild-type animals. These results suggest that C. elegans aquaporins are not essential for whole animal osmoregulation and/or that deletion of aquaporin genes activates mechanisms that compensate for loss of water channel function.
Inhibition of PI3-kinase signaling by glucocorticoids results in increased branched-chain amino acid degradation in renal epithelial cells
Authors: Wang Xiaonan , Hu Junping , Price S. Russ
Published: January 2007

Phosphatidylinositol 3-kinase(PI3K) is a pivotal enzyme involved in the control of a variety of diverse metabolic functions. Glucocorticoids have been shown to attenuate PI3K signaling in some nonrenal cell types, raising the possibility that some physiological effects of glucocorticoids in renal cells may be achieved by a similar mechanism. Therefore, we tested whether glucocorticoids affect signaling through the insulin receptor substrate (IRS)-1/PI3K/Akt signaling cascade in LLC-PK1-GR101 renal epithelial cells. Treatment of cells with dexamethasone for 24 h: 1) suppressed IRS-1-associated PI3K activity and Akt phosphorylation, 2) increased the level of the PI3K p85 regulatory subunit but not the p110 catalytic subunit, and 3) induced the phosphorylation of IRS-1 on inhibitory Ser307. We have previously reported that glucocorticoids increase branched-chain ketoacid dehydrogenase (BCKD) activity in LLC-PK1-GR101 cells. This response was achieved, in part, by alterations in the transcription of BCKD subunits and BCKD kinase, which inactivates the enzyme complex by phosphorylation. Therefore, we tested whether inhibition of PI3K signaling would mimick glucocorticoids by increasing branched-chain amino acid degradation. Expression of a dominant negative PI3K p85 regulatory subunit (Adp85ΔiSH2) increased BCKD activity, and dexamethasone did not further stimulate enzyme activity. Inhibition of PI3K using LY-294002 increased the transcription of the BCKD E2 subunit but not the E1α subunit or BCKD kinase. Thus, glucocorticoids inhibit signaling through the IRS-1/PI3K/Akt pathway with a consequence of increased branched-chain amino acid catabolism.
Assessment of renal autoregulation
Authors: Cupples William A. , Braam Branko
Published: January 2007

The kidney displays highly efficient autoregulation so that under steady-state conditions renal blood flow (RBF) is independent of blood pressure over a wide range of pressure. Autoregulation occurs in the preglomerular microcirculation and is mediated by two, perhaps three, mechanisms. The faster myogenic mechanism and the slower tubuloglomerular feedback contribute both directly and interactively to autoregulation of RBF and of glomerular capillary pressure. Multiple experiments have been used to study autoregulation and can be considered as variants of two basic designs. The first measures RBF after multiple stepwise changes in renal perfusion pressure to assess how a biological condition or experimental maneuver affects the overall pressure-flow relationship. The second uses time-series analysis to better understand the operation of multiple controllers operating in parallel on the same vascular smooth muscle. There are conceptual and experimental limitations to all current experimental designs so that no one design adequately describes autoregulation. In particular, it is clear that the efficiency of autoregulation varies with time and that most current techniques do not adequately address this issue. Also, the time-varying and nonadditive interaction between the myogenic mechanism and tubuloglomerular feedback underscores the difficulty of dissecting their contributions to autoregulation. We consider the modulation of autoregulation by nitric oxide and use it to illustrate the necessity for multiple experimental designs, often applied iteratively.
Novel role of AQP-1 in NO-dependent vasorelaxation
Authors: Herrera Marcela , Garvin Jeffrey L.
Published: January 2007

Nitric oxide (NO) produced by endothelial cells diffuses to vascular smooth muscle cells to cause dilatation of the renal vasculature and other vessels. Although it is generally assumed that NO moves from cell to cell by free diffusion, we recently showed that aquaporin-1 (AQP-1) transports NO across cell membranes. AQP-1 is expressed in endothelial and vascular smooth muscle cells. We hypothesized that diffusion of NO into vascular smooth muscle cells and out of endothelial cells is facilitated by AQP-1, and transport of NO by AQP-1 is involved in endothelium-dependent relaxation. In intact aortic rings from AQP-1 −/− mice, vasorelaxation induced by acetylcholine (which increases endogenous NO) was reduced ( P < 0.0001 vs. control). No differences were found in the relaxation caused by intracellular delivery of NO or intracellular cGMP between strains. In endothelium-denuded aortic rings from AQP-1 −/− mice, the vasorelaxant capability of NO released in the extracellular space was reduced ( P < 0.0001 vs. control). Influx of NO (5 μM) into vascular smooth muscle cells was 0.17 ± 0.02 f.u./s for control and 0.07 ± 0.01 f.u./s for AQP-1 −/− mice, 62% lower ( P < 0.002). NO released by endothelial cells in response to 1 μM acetylcholine was 96.2 ± 17.7 pmol NO/mg for control and 41.9 ± 13.4 pmol NO/mg for AQP-1 −/− mice, 56% reduction ( P < 0.04). NOS3 expression was 1.33 ± 0.29 O.D. units for control and 3.84 ± 0.76 O.D. units for AQP-1 −/− mice, 188% increase ( P < 0.01). We conclude that 1) AQP-1 facilitates NO influx into vascular smooth muscle cells, 2) AQP-1 facilitates NO diffusion out of endothelial cells, and 3) transport of NO by AQP-1 is required for full expression of endothelium-dependent relaxation.
Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator
Authors: Cheruvanky Anita , Zhou Hua , Pisitkun Trairak , Kopp Jeffrey B. , Knepper Mark A. , Yuen Peter S. T. , Star Robert A.
Published: January 2007

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10–30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at −80°C or refrigerated overnight at 4°C then stored at −80°C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.
Physical activity energy expenditure may mediate the relationship between plasma leptin levels and worsening insulin resistance independently of adiposity
Authors: Franks P. W. , Loos R. J. F. , Brage S. , O'Rahilly S. , Wareham N. J. , Ekelund U.
Published: January 2007

Leptin regulates a constellation of neuroendocrine processes that control energy homeostasis. The infusion of leptin in rodents lacking endogenous leptin promotes physical activity energy expenditure (PAEE) and improves insulin signaling, whereas hyperleptinemia is associated with physical inactivity and insulin resistance (IR). We tested whether baseline leptin levels predict changes in PAEE and IR over time, independent of obesity. We also assessed whether the relationship between leptin and change in IR is mediated by PAEE. The population consisted of 288 nondiabetic UK Caucasian adults (mean age: 49.4 yr; SD: 0.7 yr), in whom leptin, insulin, glucose, PAEE (via heart rate monitoring with individual calibration by indirect calorimetry), and anthropometric characteristics had been measured at baseline and 5 yr later. In linear regression models, baseline leptin levels inversely predicted follow-up PAEE ( P = 0.033). On average, individuals with low leptin levels (below sex-specific median) increased their daily activity 35% more during the 5-yr follow-up period than those with above-median leptin levels. Baseline leptin level also predicted worsening IR (fasting, 30-min, and 2-h insulins, and homeostasis model assessment-IR; all P < 0.01). Associations were independent of potential confounders, such as adiposity, age, and sex. Including baseline PAEE as a cofactor in the leptin-insulin models reduced the strength (1–4% reduction) and significance of the associations, suggesting that PAEE mediates the leptin-insulin relationships. Hyperleptinemia predicts a relative decline in PAEE and worsening insulin resistance, possibly via shared molecular pathways.
Cardiorespiratory and cerebrovascular responses to acute poikilocapnic hypoxia following intermittent and continuous exposure to hypoxia in humans
Authors: Ainslie Philip N. , Barach Alice , Cummings Kevin J. , Murrell Carissa , Hamlin Mike , Hellemans John
Published: January 2007

We tested the hypothesis that intermittent hypoxia (IH) and/or continuous hypoxia (CH) would enhance the ventilatory response to acute hypoxia (HVR), thereby altering blood pressure (BP) and cerebral perfusion. Seven healthy volunteers were randomly selected to complete 10–12 days of IH (5-min hypoxia to 5-min normoxia repeated for 90 min) before ascending to mild CH (1,560 m) for 12 days. Seven other volunteers did not receive any IH before ascending to CH for the same 12 days. Before the IH and CH, following 12 days of CH and 12–13 days post-CH exposure, all subjects underwent a 20-min acute exposure to poikilocapnic hypoxia (inspired fraction of O2, 0.12) in which ventilation, end-tidal gases, arterial O2 saturation, BP, and middle cerebral artery blood flow velocity (MCAV) were measured continuously. Following the IH and CH exposures, the peak HVR was elevated and was related to the increase in BP ( r = 0.66 to r = 0.88, respectively; P < 0.05) and to a reciprocal decrease in MCAV ( r = 0.73 to r = 0.80 vs. preexposures; P < 0.05) during the hypoxic test. Following both IH and CH exposures, HVR, BP, and MCAV sensitivity to hypoxia were elevated compared with preexposure, with no between-group differences following the IH and/or CH conditions, or persistent effects following 12 days of sea level exposure. Our findings indicate that IH and/or mild CH can equally enhance the HVR, which, by either direct or indirect mechanisms, facilitates alterations in BP and MCAV.
Dopamine D1/5 Receptor-Mediated Long-Term Potentiation of Intrinsic Excitability in Rat Prefrontal Cortical Neurons: Ca2+-Dependent Intracellular Signaling
Authors: Chen Long , Bohanick Joseph D. , Nishihara Makoto , Seamans Jeremy K. , Yang Charles R.
Published: January 2007

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input–output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors ( Vhold = −65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2–50 μM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 μM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297 -induced LTP-IE was significantly attenuated by Cd2+, [Ca2+]i chelation, by inhibition of phospholipase C, protein kinase-C, and Ca2+/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca2+ channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca2+ influx via non-L-type Ca2+ channels, activation of PLC, intracellular Ca2+ elevation, activation of Ca2+-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K+ and Ca2+ currents to dynamically regulate overall excitability of PFC neurons.
What Does the Brain Do When You Fake It? An fMRI Study of Pantomimed and Real Grasping
Authors: Króliczak G. , Cavina-Pratesi C. , Goodman D. A. , Culham J. C.
Published: January 2007

Given that studying neural bases of actions is very challenging with fMRI, numerous experiments have used pantomimed actions as a proxy to studying neural circuits of real actions. However, the underlying assumption that the same neural mechanisms mediate real and pantomimed actions has never been directly tested. Moreover, the assumption is called into question by neuropsychological evidence suggesting that real actions depend on the dorsal stream of visual processing whereas pretend actions also recruit the ventral stream. Here, we directly tested these ideas in neurologically intact subjects. Ten right-handed participants performed four tasks: 1) grasping real three-dimensional objects, 2) reaching toward the objects and touching them with the knuckle without hand preshaping, 3) pantomimed grasping in an adjacent location where no object was present, and 4) pantomimed reaching toward an adjacent location. As expected, in the anterior intraparietal area, there was significantly higher activation during real grasping than that during real reaching. However, the activation difference between pantomimed grasping and pantomimed reaching did not reach statistical significance. There was also no effect of pantomimed grasping within the ventral stream, including an object-selective area in the lateral occipital cortex. Instead, we found that pantomimed grasping was mediated by right-hemisphere activation, particularly the right parietal cortex. These results suggest that areas typically invoked by real actions may not necessarily be driven by “fake” actions. Moreover, pantomimed grasping may not tap object-related areas within the ventral stream, but rather may rely on mechanisms within the right hemisphere that are recruited by artificial and less practiced actions.
Reelin Signaling Facilitates Maturation of CA1 Glutamatergic Synapses
Authors: Qiu Shenfeng , Weeber Edwin J.
Published: January 2007

Reelin signaling through the low-density lipoprotein receptor family members, apoliproprotein E receptor 2 (apoER2) and very-low-density lipoprotein receptor (VLDLR), plays a pivotal role in dictating neuronal lamination during embryonic brain development. Recent evidence suggests that this signaling system also plays a role in the postnatal brain to modulate synaptic transmission, plasticity, and cognitive behavior, mostly likely due to a functional coupling with N-methyl-d-aspartate (NMDA) receptors. In this study, we investigated the effects of reelin on the maturation of CA1 glutamatergic function using electrophysiological and biochemical approaches. In cultured hippocampal slices, reelin treatment increased the amplitude of AMPAR-mediated miniature excitatory postsynaptic currents and the evoked AMPA/NMDA receptor current ratios. In addition, reelin treatment also reduced the number of silent synapses, facilitated a developmental switch from NR2B to NR2A of NMDARs, and increased surface expression of AMPARs in CA1 tissue. In cultured hippocampal neurons from reeler embryos, reduced numbers of AMPAR subunit GluR1 and NMDAR subunit NR1 clustering were observed compared with those obtained from wild-type embryos. Supplementing reelin in the reeler culture obliterated these genotypic differences. These results demonstrate that reelin- and lipoprotein receptor-mediated signaling may operate during developmental maturation of hippocampal glutamatergic function and thus represent a potential important mechanism for controlling synaptic strength and plasticity in the postnatal hippocampus.