bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonlinear Alfvén waves propagation in astrophysical plasma in the presence of dissipative effects
Authors: Ignat Margareta , Ciurea Rodica
Published: July 2002

In this paper we examine the nonlinear propagation of Alfvén waves in astrophysical plasma, taking into account the viscosity and Joule effect. Working in the second order magnetohydrodynamic (MHD) approximation, using the multiple scales method, the dispersion equation and the nonlinear Schrödinger (NLS) equation for the case in study has been obtained, emphasizing the influence of dissipative phenomena upon the interaction between the Alfvén and acoustic modes.
Dust ion-acoustic shocks and holes
Authors: Shukla P. K.
Published: July 2002

Properties of dust ion-acoustic (DIA) shocks and DIA holes are examined, assuming that a dusty plasma contains immobile charged dust grains, warm electrons, and warm ions. A Kortweg-deVries- Burgers equation for DIA shocks is derived by employing the hydrodynamic equations for the warm ion fluid and the Boltzmann electron density distribution. On the other hand, DIA holes arise when the warm ions follow a trapped vortex distribution. The profiles of DIA shocks and DIA holes are discussed. The results are useful for understanding the salient features of DIA shocks and holes that are recently observed in laboratory dusty plasma devices.
Stability of solitary kinetic Alfvén waves and ion-acoustic waves in a nonthermal plasma
Authors: Bandyopadhyay Anup , Das K. P.
Published: July 2002

The KdV-ZK (Korteweg–de Vries–Zakharov–Kuznetsov) equations for kinetic Alfvén and ion-acoustic waves in a nonthermal magnetized plasma have been derived. The coefficient of the nonlinear term of this equation for an ion-acoustic wave can vanish along a curve in a two-dimensional parameter space. In this case, two coupled equations constituting a modified KdV-ZK equation describing nonlinear behavior of an ion-acoustic wave are derived. The solitary wave solutions of all these equations are obtained and their stabilities are investigated by the Rowlands–Infeld method. It is found that the kinetic Alfvén solitons are stable. The instability conditions and the maximum growth rate of the instability for ion-acoustic solitons are determined.
Large aperture point-focusing diffractor for x rays
Authors: Wittry D. B. , Golijanin D. M.
Published: July 2002

A doubly bent germanium single crystal with toroidally curved crystal planes was used to obtain three-dimensional focusing of monochromatic x rays from a microfocus source. The Johansson geometry in radial planes was obtained by cutting a cylindrically curved lamella, polishing, etching, and bending over a spherical mold of ∼50 mm radius at 740 °C. The bent crystal was aligned to obtain the Cu Kα (333) reflection in the spot which had a principal focus of ∼100 μm. Allowing for anode pitting and extraneous reflections, the observed x-ray intensity was ∼10 times lower than expected theoretically. The discrepancy was attributed to imperfect bending and crystal defects.
Selective induction of neuropilin-1 by vascular endothelial growth factor (VEGF): A mechanism contributing to VEGF-induced angiogenesis
Authors: Oh Hideyasu , Takagi Hitoshi , Otani Atsushi , Koyama Shinji , Kemmochi Seiji , Uemura Akiyoshi , Honda Yoshihito
Published: July 2002

Neuropilin (NRP) 1, previously identified as a neuronal
receptor that mediates repulsive growth cone guidance, has been shown
recently to function also in endothelial cells as an isoform-specific
receptor for vascular endothelial growth factor (VEGF)
165
and as a coreceptor
in vitro
of VEGF receptor 2.
However, its potential role in pathologic angiogenesis remains unknown.
In the present study, we first show that VEGF selectively up-regulates
NRP1 but not NRP2 via the VEGF receptor 2-dependent pathway. By NRP1
binding analysis, we showed that its induction by VEGF accompanies
functional receptor expression. Endothelial proliferation stimulated by
VEGF
165
was inhibited significantly by antibody
perturbation of NRP1. In a murine model of VEGF-dependent
angioproliferative retinopathy, intense NRP1 mRNA expression was
observed in the newly formed vessels. Furthermore, selective NRP1
inhibition in this model suppressed neovascular formation
substantially. These results suggest that VEGF cannot only activate
endothelial cells directly but also can contribute to robust
angiogenesis
in vivo
by a mechanism that involves
up-regulation of its cognate receptor expression.
Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes
Authors: Liou Yih-Cherng , Ryo Akihide , Huang Han-Kuei , Lu Pei-Jung , Bronson Roderick , Fujimori Fumihiro , Uchida Takafumi , Hunter Tony , Lu Kun Ping
Published: July 2002

Phosphorylation of proteins on serine/threonine residues preceding proline is a key signaling mechanism. The conformation and function of a subset of these phosphorylated proteins is regulated by the prolyl isomerase Pin1 through isomerization of phosphorylated Ser/Thr-Pro bonds. Although young Pin1
−/−
mice have been previously shown to develop normally, we show here that they displayed a range of cell-proliferative abnormalities, including decreased body weight and testicular and retinal atrophies. Furthermore, in Pin1
−/−
adult females, the breast epithelial compartment failed to undergo the massive proliferative changes associated with pregnancy. Interestingly, many of these Pin1-deficient phenotypes such as retinal hypoplasia and mammary gland impairment are also the characteristic of cyclin D1-deficient mice. Cyclin D1 levels were significantly reduced in many tissues in Pin1-deficient mice, including retina and breast epithelial cells from pregnant mice. Moreover, Pin1 directly bound to cyclin D1 phosphorylated on Thr-286–Pro increased cyclin D1 in the nucleus and stabilized cyclin D1. These results indicate that Pin1 positively regulates cyclin D1 function at the transcriptional level, as demonstrated previously, and also through posttranslational stabilization, which together explain why Pin1 loss-of-function phenotypes in the mouse resemble cyclin D1-null phenotypes. Our results provide genetic evidence for an essential role of Pin1 in maintaining cell proliferation and regulating cyclin D1 function.
QscR, a modulator of quorum-sensing signal synthesis and virulence in
Pseudomonas aeruginosa
Authors: Chugani Sudha A. , Whiteley Marvin , Lee Kimberly M. , D'Argenio David , Manoil Colin , Greenberg E. P.
Published: July 2002

The opportunistic pathogenic bacterium
Pseudomonas
aeruginosa
uses quorum-sensing signaling systems as global
regulators of virulence genes. There are two quorum-sensing signal
receptor and signal generator pairs, LasR–LasI and RhlR–RhlI. The
recently completed
P. aeruginosa
genome-sequencing
project revealed a gene coding for a homolog of the signal receptors,
LasR and RhlR. Here we describe a role for this gene, which we call
qscR
. The
qscR
gene product governs the
timing of quorum-sensing-controlled gene expression and it dampens
virulence in an insect model. We present evidence that suggests the
primary role of QscR is repression of
lasI
. A
qscR
mutant produces the LasI-generated signal
prematurely, and this results in premature transcription of a number of
quorum-sensing-regulated genes. When fed to
Drosophila
melanogaster
, the
qscR
mutant kills the animals
more rapidly than the parental
P. aeruginosa
. The
repression of
lasI
by QscR could serve to ensure that
quorum-sensing-controlled genes are not activated in environments where
they are not useful.
Microrheometry of semiflexible actin networks through enforced single-filament reptation: Frictional coupling and heterogeneities in entangled networks
Authors: Dichtl M. A. , Sackmann E.
Published: July 2002

Magnetic tweezers are applied to study the enforced motion of single actin filaments in entangled actin networks to gain insight into friction-mediated entanglement in semiflexible macromolecular networks. Magnetic beads are coupled to one chain end of test filaments, which are pulled by 5 to 20 pN force pulses through entangled solutions of nonlabeled actin, the test filaments thus acting as linear force probes of the network. The transient filament motion is analyzed by microfluorescence, and the deflection-versus-time curves of the beads are evaluated in terms of a mechanical equivalent circuit to determine viscoelastic parameters, which are then interpreted in terms of viscoelastic moduli of the network. We demonstrate that the frictional coefficient characterizing the hydrodynamic coupling of the filaments to the surrounding network is much higher than predicted by the tube model, suggesting that friction-mediated interfilament coupling plays an important role in the entanglement of non-cross-linked actin networks. Furthermore, the local tube width along the filament contour (measured in terms of the root-mean-square displacement characterizing the lateral Brownian motion of the test filament) reveals strong fluctuations that can lead to transient local pinching of filaments.
Bombesin antagonists inhibit growth of MDA-MB-435 estrogen-independent breast cancers and decrease the expression of the ErbB-2/HER-2 oncoprotein and c-
jun
and c-
fos
oncogenes
Authors: Bajo Ana M. , Schally Andrew V. , Krupa Magdalena , Hebert Francine , Groot Kate , Szepeshazi Karoly
Published: July 2002

Previous studies showed that antagonists of bombesin (BN)/gastrin-releasing peptide (GRP) inhibit the growth of various cancers by interfering with the growth-stimulatory effects of BN-like peptides and down-regulating epidermal growth factor receptors on tumors. Because the overexpression of the human epidermal growth factor receptor-2 (ErbB-2/HER-2/neu) oncogene plays a role in the progression of many breast cancers, we investigated whether BN/GRP antagonists can affect HER-2 in mammary tumors. Female nude mice bearing orthotopic xenografts of MDA-MB-435 human estrogen-independent breast cancers were treated daily with BN/GRP antagonists RC-3095 (20 μg) or RC-3940-II (10 μg) for 6 weeks. The expression of BN/GRP receptors on tumors was analyzed by reverse transcription–PCR and immunoblotting. We also evaluated whether the mRNA expression for the c-
jun
and c-
fos
oncogenes is affected by the therapy. Both BN/GRP antagonists significantly inhibited growth of MDA-MB-435 cancers; RC-3095 reduced tumor volume by 40% and RC-3940-II by 65%. The GRP receptors (subtype 1) were detected in MDA-MB-435 tumors, showing that they mediate the inhibitory effect of the antagonists. Tumor inhibition was associated with a substantial reduction in the expression of mRNA and protein levels of the ErbB/HER receptor family as well as with a decrease in the expression of c-
jun
and c-
fos
oncogenes. BN/GRP antagonists RC-3940-II and RC-3095 could be considered for endocrine therapy of estrogen-independent breast cancers that express members of the ErbB/HER receptor family and the c-
jun
and c-
fos
oncogenes.
Molecular mechanisms of cocaine reward: Combined dopamine and serotonin transporter knockouts eliminate cocaine place preference
Authors: Sora Ichiro , Hall F. Scott , Andrews Anne M. , Itokawa Masanari , Li Xiao-Fei , Wei Hong-Bing , Wichems Christine , Lesch Klaus-Peter , Murphy Dennis L. , Uhl George R.
Published: July 2002

Cocaine blocks uptake by neuronal plasma membrane
transporters for dopamine (DAT), serotonin (SERT), and norepinephrine
(NET). Cocaine reward/reinforcement has been linked to actions at DAT
or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET
reduce cocaine reward/reinforcement, leaving substantial uncertainty
about cocaine's molecular mechanisms for reward. Conceivably, the
molecular bases of cocaine reward might display sufficient redundancy
that either DAT or SERT might be able to mediate cocaine reward in the
other's absence. To test this hypothesis, we examined double knockout
mice with deletions of one or both copies of both the DAT and SERT
genes. These mice display viability, weight gain, histologic features,
neurochemical parameters, and baseline behavioral features that allow
tests of cocaine influences. Mice with even a single wild-type DAT gene
copy and no SERT copies retain cocaine reward/reinforcement, as
measured by conditioned place-preference testing. However, mice with no
DAT and either no or one SERT gene copy display no preference for
places where they have previously received cocaine. The serotonin
dependence of cocaine reward in DAT knockout mice is thus confirmed by
the elimination of cocaine place preference in DAT/SERT double
knockout mice. These results provide insights into the brain molecular
targets necessary for cocaine reward in knockout mice that develop in
their absence and suggest novel strategies for anticocaine medication
development.
Looped out and perpendicular: Deformation of Watson–Crick base pair associated with actinomycin D binding
Authors: Chou Shan-Ho , Chin Ko-Hsin , Chen Fu-Ming
Published: July 2002

Many anticancer drugs interact directly with DNA to exert their biological functions. To date, all noncovalent, intercalating drugs interact with DNA exclusively by inserting their chromophores into base steps to form elongated and unwound duplex structures without disrupting the flanking base pairs. By using actinomycin D (ActD)-5′-GXC/CYG-5′ complexes as examples, we have found a rather unusual interaction mode for the intercalated drug; the central Watson–Crick X/Y base pairs are looped out and displaced by the ActD chromophore. The looped-out bases are not disordered but interact perpendicularly with the base/chromophore and form specific H bonds with DNA. Such a complex structure provides intriguing insights into how ligand interacts with DNA and enlarges the repertoires for sequence-specific DNA recognition.
Reproductive constraints and the evolution of life histories with indeterminate growth
Authors: Charnov Eric L. , Turner Thomas F. , Winemiller Kirk O.
Published: July 2002

A prominent feature of comparative life histories in fish (and other indeterminate growers) is the approximate invariance across species of certain dimensionless numbers made up from reproductive and timing variables. The two best known are the age at maturity (α) divided by the average adult lifespan (E), and the proportion of a body mass given to reproduction per year (c) multiplied byE. This article uses evolutionary life-history theory for nongrowing populations to predict the numeric value of these numbers for fish and lizards, with several new implications for the dynamics of ontogenetic development.
Molecular characterization and assembly of the needle complex of the
Salmonella typhimurium
type III protein secretion system
Authors: Kubori Tomoko , Sukhan Anand , Aizawa Shin-Ichi , Galán Jorge E.
Published: July 2002

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The
Salmonella typhimurium
pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.
UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II
Authors: Rockx Davy A. P. , Mason Rebecca , van Hoffen Anneke , Barton Michelle Craig , Citterio Elisabetta , Bregman David B. , van Zeeland Albert A. , Vrieling Harry , Mullenders Leon H. F.
Published: July 2002

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an
in vitro
transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged β-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.
HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase
Authors: Espeseth Amy S. , Felock Peter , Wolfe Abigail , Witmer Marc , Grobler Jay , Anthony Neville , Egbertson Melissa , Melamed Jeffrey Y. , Young Steve , Hamill Terence , Cole James L. , Hazuda Daria J.
Published: July 2002

Diketo acids such as L-731,988 are potent inhibitors of HIV-1
integrase that inhibit integration and viral replication in cells.
These compounds exhibit the unique ability to inhibit the strand
transfer activity of integrase in the absence of an effect on 3′ end
processing. To understand the reasons for this distinct inhibitory
profile, we developed a scintillation proximity assay that permits
analysis of radiolabeled inhibitor binding and integrase function.
High-affinity binding of L-731,988 is shown to require the assembly of
a specific complex on the HIV-1 long terminal repeat. The interaction
of L-731,988 with the complex and the efficacy of L-731,988 in strand
transfer can be abrogated by the interaction with target substrates,
suggesting competition between the inhibitor and the target DNA. The
L-731,988 binding site and that of the target substrate are thus
distinct from that of the donor substrate and are defined by a
conformation of integrase that is only adopted after assembly with the
viral end. These results elucidate the basis for diketo acid inhibition
of strand transfer and have implications for integrase-directed HIV-1
drug discovery efforts.
IL-1β-converting enzyme (caspase-1) in intestinal inflammation
Authors: Siegmund Britta , Lehr Hans-Anton , Fantuzzi Giamila , Dinarello Charles A.
Published: July 2002

IL-1β-converting enzyme (ICE; caspase-1) is the intracellular protease that cleaves the precursors of IL-1β and IL-18 into active cytokines. In the present study, the effect of ICE deficiency was evaluated during experimental colitis in mice. In acute dextran sulfate sodium-induced colitis, ICE-deficient (ICE KO) mice exhibited a greater than 50% decrease of the clinical scores weight loss, diarrhea, rectal bleeding, and colon length, whereas daily treatment with IL-1 receptor antagonist revealed a modest reduction in colitis severity. To further characterize the function of ICE and its role in intestinal inflammation, chronic colitis was induced over a 30-day time period. During this chronic time course, ICE KO mice exhibited a near complete protection, as reflected by significantly reduced clinical scores and almost absent histological signs of colitis. Consistently, colon shortening occurred only in dextran sulfate sodium-exposed wild-type mice but not in ICE KO mice. Protection was accompanied by reduced spontaneous release of the proinflammatory cytokines IL-18, IL-1β, and IFN-γ from total colon cultures. In addition, flow cytometric analysis of isolated mesenteric lymph node cells revealed evidence of reduced cell activation in ICE KO mice as evaluated by surface expression of CD3 CD69 and CD4 CD25. We conclude that inhibition of ICE represents a novel anti-inflammatory strategy for intestinal inflammation.
Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis
Authors: Jiang Aixiang , Lehti Kaisa , Wang Xing , Weiss Stephen J. , Keski-Oja Jorma , Pei Duanqing
Published: July 2002

Membrane-type matrix metalloproteinase 1 (MT1-MMP) plays a critical role in extracellular matrix remodeling under both physiological and pathological conditions. However, the mechanisms controlling its activity on the cell surface remain poorly understood. In this study, we demonstrate that MT1-MMP is regulated by endocytosis. First, we determined that Con A induces proMMP-2 activation in HT1080 cells by shifting endogenous MT1-MMP from intracellular compartments to cell surface. This phenotype was mimicked by the cytoplasmic truncation mutant MT1ΔC with more robust pro-MMP-2 activation and cell surface expression than wild-type MT1-MMP in transfected cells. MT1ΔC was subsequently shown to be resistant to Con A treatment whereas MT1-MMP remains competent, suggesting that Con A regulates MT1-MMP activity through cytoplasmic domain-dependent trafficking. Indeed, MT1-MMP was colocalized with clathrin on the plasma membrane and with endosomal antigen 1 in endosomes. Internalization experiments revealed that MT1-MMP is internalized rapidly in clathrin-coated vesicles whereas MT1ΔC remains on cell surface. Coexpression of a dominant negative mutant of dynamin, K44A, resulted in elevation of MT1-MMP activity by interfering with the endocytic process. Thus, MT1-MMP is regulated by dynamin-dependent endocytosis in clathrin-coated pits through its cytoplasmic domain.
Host cell factor requirement for hepatitis C virus enzyme maturation
Authors: Waxman Lloyd , Whitney Michael , Pollok Brian A. , Kuo Lawrence C. , Darke Paul L.
Published: July 2002

The cellular chaperone, HSP90, is identified here as an essential factor for the activity of NS2/3 protease of hepatitis C virus. The cleavage activity of NS2/3 protease synthesized in reticulocyte lysate is ATP-dependent, as evidenced by ATP depletion experiments and inhibition with nonhydrolyzable ATP analogs. Geldanamycin and radicicol, ATP-competitive inhibitors of the chaperone HSP90, also inhibit the cleavage of
in vitro
-synthesized NS2/3. Furthermore, these HSP90 inhibitors prevent NS2/3 cleavage when the protease is expressed in mammalian cells. The physical association of NS2/3 with HSP90 is demonstrated by immunoprecipitation. Thus, by way of a chaperone/folding activity, an HSP90-containing complex is required for maturation of the polyprotein that encodes the enzymes essential for hepatitis C virus replication.
The dissociation of acetylcholine from open nicotinic receptor channels
Authors: Grosman Claudio , Auerbach Anthony
Published: July 2002

Ligand-gated ion channels bind agonists with higher affinity in the open than in the closed state. The kinetic basis of this increased affinity has remained unknown, because even though the rate constants of agonist association to and dissociation from closed receptors can be estimated with reasonable certainty, the kinetics of the binding steps in
open
receptors have proven to be elusive. To be able to measure the agonist-dissociation rate constant from open muscle nicotinic receptors, we increased the probability of ligand unbinding from the open state by engineering a number of mutations that speed up opening and slow down closing but leave the ligand-binding properties unchanged. Single-channel patch-clamp recordings from the wild-type and mutant constructs were performed at very low concentrations of acetylcholine (ACh). The durations of individual channel activations were analyzed assuming that “bursts” of fully liganded (diliganded) receptor openings can be terminated by ligand dissociation from the closed or open state (followed by fast closure) or by desensitization. This analysis revealed that ACh dissociates from diliganded open receptors at ≈24 s
−1
, that is, ≈2,500 times more slowly than from diliganded closed receptors. This change alone without a concomitant change in the association rate constant to the open state quantitatively accounts for the increased equilibrium affinity of the open channel for ACh. Also, the results predict that both desensitization and ACh dissociation from the open state frequently terminate bursts of openings in naturally occurring gain-of-function mutants (which cause slow-channel congenital myasthenia) and therefore would contribute significantly to the time course of the endplate current decay in these disease conditions.
Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells
Authors: Neves Guilherme , Gomis Ana , Lagnado Leon
Published: July 2002

To investigate the regulation of endocytosis by Ca
2+
,
we have made capacitance measurements in the synaptic terminal of
depolarizing bipolar cells from the retina of goldfish. After a
brief depolarization, all of the excess membrane was retrieved rapidly
(τ ≈1 s). But when the rise in free [Ca
2+
] was
reduced by the introduction of Ca
2+
buffers
[1,2-bis(2-aminophenoxy)ethane-
N,N,N
′
,N
′-tetraacetate
(BAPTA) or EGTA], a large fraction of the membrane was retrieved by a
second, slower mechanism (τ ≥ 10 s). The block of fast
endocytosis by EGTA could be overcome by increasing the amplitude of
the Ca
2+
current, demonstrating that Ca
2+
influx was the trigger for fast endocytosis. These manipulations of the
Ca
2+
signal altered the relative proportions of fast and
slow endocytosis but did not modulate the rate constants of these
processes. A brief stimulus that triggered fast endocytosis did not
generate a significant rise in the spatially averaged
[Ca
2+
], indicating that Ca
2+
regulated
endocytosis through an action close to the active zone. The slow mode
of retrieval occurred at the resting [Ca
2+
]. These
results demonstrate that Ca
2+
influx couples fast
endocytosis and exocytosis at this synapse.
The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness
Authors: Humbles Alison A. , Lu Bao , Friend Daniel S. , Okinaga Shoji , Lora Jose , Al-garawi Amal , Martin Thomas R. , Gerard Norma P. , Gerard Craig
Published: July 2002

CCR3 is a chemokine receptor initially thought specific to eosinophils but subsequently identified on TH2 cell subsets, basophils, mast cells, neural tissue, and some epithelia. Because of the prominent role of these cells in allergic disease, including asthma, we generated mice deficient in CCR3 to determine its contribution in a model of allergic airway disease. Here we show that CCR3 is important for the basal trafficking of eosinophils to the intestinal mucosa but not the lung. In contrast, CCR3 disruption significantly curtails eosinophil recruitment to the lung after allergen challenge, with the majority of the eosinophils being arrested in the subendothelial space. Further, a role for CCR3 in mast cell homing has been identified; after sensitization and allergen challenge, we find increased numbers of intraepithelial mast cells in the trachea of knockout mice. Physiologically, we find that the net result of these complex cell fates after sensitization and allergen challenge is a paradoxical increase in airway responsiveness to cholinergic stimulation. These data underscore a more complex role for CCR3 in allergic disease than was anticipated.
CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia
in vitro
and in mouse nasal mucosa
in vivo
Authors: Ostedgaard Lynda S. , Zabner Joseph , Vermeer Daniel W. , Rokhlina Tatiana , Karp Philip H. , Stecenko Arlene A. , Randak Christoph , Welsh Michael J.
Published: July 2002

In developing gene therapy for cystic fibrosis (CF) airways disease, a transgene encoding a partially deleted CF transmembrane conductance regulator (CFTR) Cl
−
channel could be of value for vectors such as adeno-associated virus that have a limited packaging capacity. Earlier studies in heterologous cells indicated that the CFTR R (regulatory) domain is predominantly random coil and that parts of the R domain can be deleted without abolishing channel function. Therefore, we designed a series of CFTR variants with shortened R domains (between residues 708 and 835) and expressed them in well-differentiated cultures of CF airway epithelia. All of the variants showed normal targeting to the apical membrane, and for the constructs we tested, biosynthesis was like wild type. Moreover, all constructs generated transepithelial Cl
−
current in CF epithelia. Comparison of the Cl
−
transport suggested that the length of the R domain, the presence of phosphorylation sites, and other factors contribute to channel activity. A variant deleting residues 708–759 complemented CF airway epithelia to the same extent as wild-type CFTR and showed no current in the absence of cAMP stimulation. In addition, expression in nasal mucosa of CF mice corrected the Cl
−
transport defect. These data provide insight into the structure and function of the R domain and identify regions that can be deleted with retention of function. Thus they suggest a strategy for shortening the transgene used in CF gene therapy.
Identification, sequence, and expression of caveolin-2 defines a caveolin gene family.
Authors: Scherer P E , Okamoto T , Chun M , Nishimoto I , Lodish H F , Lisanti M P
Published: July 2002

Caveolin, a 21- to 24-kDa integral membrane protein, is a principal component of caveolae membranes. Caveolin interacts directly with heterotrimeric guanine nucleotide binding proteins (G proteins) and can functionally regulate their activity. Here, an approximately 20-kDa caveolin-related protein, caveolin-2, was identified through microsequencing of adipocyte-derived caveolin-enriched membranes; caveolin was retermed caveolin-1. Caveolins 1 and 2 are similar in most respects. mRNAs for both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during adipocyte differentiation. Caveolin-2 colocalizes with caveolin-1, indicating that caveolin-2 also localizes to caveolae. However, caveolin-1 and caveolin-2 differ in their functional interactions with heterotrimeric G proteins, possibly explaining why caveolin-1 and -2 are coexpressed within a single cell.
Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.
Authors: Collins P L , Hill M G , Cristina J , Grosfeld H
Published: July 2002

RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions.
High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation.
Authors: Grohovaz F , Bossi M , Pezzati R , Meldolesi J , Tarelli F T
Published: July 2002

We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze-drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.
Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene.
Authors: Gladyshev V N , Jeang K T , Stadtman T C
Published: July 2002

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenium metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)-containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminal-deduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val-Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved C-terminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system.
Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.
Authors: Cruz-Reyes J , Sollner-Webb B
Published: July 2002

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.
Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.
Authors: Hirose K , Lockhart A , Cross R A , Amos L A
Published: July 2002

Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single- and double-headed kinesin and double-headed ncd. Three-dimensional density maps and difference maps show that, in adenosine 5'-[beta,gamma-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ncd heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ncd points toward the minus end.
Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications.
Authors: Powell J F , Reska-Skinner S M , Prakash M O , Fischer W H , Park M , Rivier J E , Craig A G , Mackie G O , Sherwood N M
Published: July 2002

The neuropeptide gonadotropin-releasing hormone (GnRH) is the major regulator of reproduction in vertebrates. Our goal was to determine whether GnRH could be isolated and identified by primary structure in a protochordate and to examine its location by immunocytochemistry. The primary structure of two novel decapeptides from the tunicate Chelyosoma productum (class Ascidiacea) was determined. Both show significant identity with vertebrate GnRH. Tunicate GnRH-I (pGlu-His-Trp-Ser-Asp-Tyr-Phe-Lys-Pro-Gly-NH2) has 60% of its residues conserved, compared with mammalian GnRH, whereas tunicate GnRH-II (pGlu-His-Trp-Ser-Leu-Cys-His-Ala-Pro-Gly-NH2) is unusual in that it was isolated as a disulfide-linked dimer. Numerous immunoreactive GnRH neurons lie within blood sinuses close to the gonoducts and gonads in both juveniles and adults, implying that the neuropeptide is released into the bloodstream. It is suggested that in ancestral chordates, before the evolution of the pituitary, the hormone was released into the bloodstream and acted directly on the gonads.
Induction of "tissue" transglutaminase in HIV pathogenesis: evidence for high rate of apoptosis of CD4+ T lymphocytes and accessory cells in lymphoid tissues.
Authors: Amendola A , Gougeon M L , Poccia F , Bondurand A , Fesus L , Piacentini M
Published: July 2002

Peripheral blood mononuclear cells and lymphoid tissues from HIV-infected individuals display high levels of "tissue" transglutaminase (tTG) with respect to seronegative persons. In asymptomatic individuals, > 80% of the circulating CD4+ T cells synthesize tTG protein and the number of these cells matches the level of apoptosis detected in the peripheral blood mononuclear cells from the same patients. In HIV-infected lymph nodes tTG protein is localized in large number of cells (macrophages, follicular dendritic cells, and endothelial cells), showing distinctive morphological and biochemical features of apoptosis as well as in lymphocytes and syncytia. These findings demonstrate that during the course of HIV infection, high levels of apoptosis also occur in the accessory cells of lymphoid organs. The increased concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the degradation product of tTG cross-linked proteins, observed in the blood of HIV-infected individuals demonstrates that the enzyme accumulated in the dying cells actively cross-links intracellular proteins. The enhanced levels of epsilon(gamma-glutamyl)lysine in the blood parallels the progression of HIV disease, suggesting that the isodipeptide determination might be a useful method to monitor the in vivo rate of apoptosis.
Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family.
Authors: Orlandini M , Marconcini L , Ferruzzi R , Oliviero S
Published: July 2002

Using a mRNA differential screening of fibroblasts differing for the expression of c-fos we isolated a c-fos-induced growth factor (FIGF). The deduced protein sequence predicts that the cDNA codes for a new member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. Northern blot analysis shows that FIGF expression is strongly reduced in c-fos-deficient cells. Transfection of exogenous c-fos driven by a constitutive promoter restores the FIGF expression in these cells. In contrast, both PDGF and VEGF expression is unaffected by c-fos. FIGF is a secreted dimeric protein able to stimulate mitogenic activity in fibroblasts. FIGF overexpression induces morphological alterations in fibroblasts. The cells acquire a spindle-shaped morphology, become more refractive, disorganized, and detach from the plate. These results imply that FIGF is a downstream growth and morphogenic effector of c-fos. These results also suggest that the expression of FIGF in response to c-fos activation induces specific differentiation patterns and its aberrant activation contributes to the malignant phenotype of tumors.
Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukemogenesis.
Authors: Rhoades K L , Hetherington C J , Rowley J D , Hiebert S W , Nucifora G , Tenen D G , Zhang D E
Published: July 2002

AML1 is involved in the (8;21) translocation, associated with acute myelogenous leukemia (AML)-type M2, which results in the production of the AML1-ETO fusion protein: the amino-terminal 177 amino acids of AML1 and the carboxyl-terminal 575 amino acids of ETO. The mechanism by which AML1-ETO accomplishes leukemic transformation is unknown; however, AML1-ETO interferes with AML1 transactivation of such AML1 targets as the T-cell receptor beta enhancer and the granulocyte-macrophage colony-stimulating factor promoter. Herein, we explored the effect of AML1-ETO on regulation of a myeloid-specific AML1 target, the macrophage colony-stimulating factor (M-CSF) receptor promoter. We found that AML1-ETO and AML1 work synergistically to transactivate the M-CSF receptor promoter, thus exhibiting a different activity than previously described. Truncation mutants within the ETO portion of AML1-ETO revealed the region of ETO necessary for the cooperativity between AML1 and AML1-ETO lies between amino acids 347 and 540. Endogenous M-CSF receptor expression was examined in Kasumi-1 cells, derived from a patient with AML-M2 t(8;21) and the promonocytic cell line U937. Kasumi-1 cells exhibited a significantly higher level of M-CSF receptor expression than U937 cells. Bone marrow from patients with AML-M2 t(8;21) also exhibited a higher level of expression of M-CSF receptor compared with normal controls. The upregulation of M-CSF receptor expression by AML1-ETO may contribute to the development of a leukemic state in these patients.
Long-range disruption of gene expression by a selectable marker cassette
Authors: Pham Christine T. N. , MacIvor Debra M. , Hug Bruce A. , Heusel Jonathan W. , Ley Timothy J.
Published: July 2002

Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.