bluuebunny/crossref_metadata_2025 huggingface dataset:
Irrigated‐Tip Catheter Ablation of Pulmonary Veins for Treatment of Atrial Fibrillation
Authors: MACLE LAURENT , JAÏS PIERRE , WEERASOORIYA RUKSHEN , HOCINI MÉLÈZE , SHAH DIPEN C. , CHOI KEE‐JOON , SCAVÉE CHRISTOPHE , RAYBAUD FLORENCE , CLÉMENTY JACQUES , HAÏSSAGUERRE MICHEL
Published: August 2003

Irrigated‐Tip Catheter Ablation of PVs.
Introduction: Catheter ablation of pulmonary veins (PV) for treatment of atrial fibrillation (AF) is limited by the disparate requirements of sufficient energy delivery to achieve PV isolation while avoiding PV stenosis. The aim of the present study was to evaluate the safety and efficacy of using an irrigated‐tip catheter for systematic isolation of PV.

Methods and Results: The study population consisted of 136 consecutive patients (109 men, mean age 52 ± 10 years) with symptomatic, drug‐refractory paroxysmal (122) or persistent (14) AF. Cavotricuspid isthmus ablation and systematic radiofrequency isolation of all four PVs (guided by a circumferential mapping catheter) was performed in all patients with a protocol using an irrigated‐tip catheter. PV diameter was assessed by selective angiography. The electrophysiologic endpoint of PV isolation was achieved in 100% of patients. Bidirectional cavotricuspid isthmus block was achieved in 99% of patients. Moderate PV stenosis (50% narrowing) was observed in one patient (0.7%) without clinical consequence. No other complications were observed. Reablation procedures were required in 67 patients (49%). After a mean follow‐up of 8.8 ± 5.3 months, 81% of patients were free of AF clinical recurrence, including 66% not taking any antiarrhythmic drugs.

Conclusion: Systematic radiofrequency ablation of PV using an irrigated‐tip catheter in patients with atrial fibrillation allows complete isolation of all four PVs with a very low incidence of stenosis.
Acute Results of Transvenous Cryoablation of Supraventricular Tachycardia (Atrial Fibrillation, Atrial Flutter, Wolff‐Parkinson‐White Syndrome, Atrioventricular Nodal Reentry Tachycardia)
Authors: RODRIGUEZ LUZ‐MARIA , GELLER J. CHRISTOPH , TSE HUNG‐FAT , TIMMERMANS CARL , REEK SVEN , LEE KATHY LAI‐FUN , AYERS GREGORY M. , LAU CHU‐PAK , KLEIN HELMUT U. , CRIJNS HARRY J.G.M
Published: August 2003

Transvenous Cryoablation for SVT.
Introduction: Radiofrequency (RF) catheter ablation currently is used for treatment of cardiac arrhythmias. Although the success rate is high for almost all supraventricular tachycardias (SVT), this technique has some drawbacks, especially when pulmonary veins (PV) are targeted for treatment of atrial fibrillation (AF). Additionally, new techniques for isolation of the PVs have the drawback that they can be used only for PV isolation and not for routine treatment of other SVTs. The aim of this study was to report on the safety and efficacy of a new cryoablation system for treatment of all SVTs.

Methods and Results: Forty‐nine patients with SVT (38 men; age 48 years, range 23–76) were enrolled in the study. Five patients were withdrawn from the study before they underwent cryoablation. The remaining 44 patients were treated with cryoablation (22 AF, 15 atrial flutter, 3 accessory pathway, 2 AV nodal reentrant tachycardia, 1 AV junction ablation for permanent AF, 1 atrial tachycardia). Cryoablation was performed with the CryoCorTM cryoablation system, which uses a precooling system and N2O as a refrigerant. The number of freezes applied varied according to the index arrhythmia treated. Successful isolation of PVs was performed in 20 of (96%) 21 AF patients and 53 of 55 veins. The overall acute success was 98% (43/44). Fifty‐three PVs were isolated (2.5/patient). The success rate was 100% (23/23) for right‐sided procedures. The average and nadir temperatures reached in right‐sided and left‐sided procedures were −77°C and −80°C and −75°C and −78°C, respectively. No acute PV stenosis was seen.

Conclusion: This novel cryoablation system appears to be safe and can successfully treat different types of SVTs, including AF. Isolation of PVs is possible without producing stenosis. Despite the high blood flow in the right atrial isthmus and PVs, bidirectional conduction block can be achieved.
Interatrial Transseptal Electrical Conduction: Comparison of Patients with Atrial Fibrillation and Normal Controls
Authors: O'DONNELL DAVID , BOURKE JOHN P. , FURNISS STEVE S.
Published: August 2003

Interatrial Electrical Conduction.
Introduction: This study analyzed the electrophysiologic properties of interatrial transseptal electrical conduction at Bachmann's bundle and the ostium of the coronary sinus (CS os) in response to pulmonary vein (PV) stimuli, which mimicked spontaneous ectopy.

Methods and Results: Forty patients with atrial fibrillation (AF) referred for ablation (15 persistent AF and 25 paroxysmal AF) and 15 control patients were enrolled in the study. During decremental extrastimulus pacing from the PVs, right atrial activation was analyzed using noncontact mapping and multipolar catheters. The refractory periods and conduction times were calculated for Bachmann's bundle, CS os, and left atrium. The dispersion of refractoriness was calculated as the difference between the refractory periods of Bachmann's bundle and the CS os. The refractory period at Bachmann's bundle was 244 msec in the persistent AF group, 213 msec in the paroxysmal AF group, and 199 msec for controls. The refractory period at the CS os was 220 msec in the persistent AF group, 201 msec in the paroxysmal AF group, and 193 msec for controls. The dispersion of refractoriness was 54 msec in the persistent AF group; this was significantly greater than in paroxysmal AF at 32 msec (P < 0.05) and controls at 13 msec (P < 0.01). During decremental pacing, lengthening of conduction times at both Bachmann's bundle and the CS os were significantly greater in the persistent AF group compared with paroxysmal AF or control groups. A higher dispersion of refractoriness was associated with a higher incidence of inducible AF and a lower rate of ablation success.

Conclusion: There are differences between the left to right interatrial electrical connections between patients with persistent AF, paroxysmal AF and controls.
Is it Safe to Program a Long Tachycardia Detection Interval?
Authors: KÜHLKAMP VOLKER , DÖRNBERGER VOLKER , BOSCH RALPH R. , KETTERING KLAUS , MEWIS CHRISTIAN
Published: August 2003

Safety of Detection in an ICD. Implantable cardioverter defibrillator (ICD) therapy is used frequently in patients with “slow” ventricular tachycardia (VT). Hence, the tachycardia detection interval is programmed within the range of the physiologic heart rate, but this may cause serious problems. If a fast VT is converted to sinus tachycardia with a cycle length shorter than the tachycardia detection interval, the episode is not terminated and the success counter is not reset. If this happens repeatedly, therapies will be exhausted without termination of the episode. If VT recurs within such an episode, it will not be treated. This report describes a patient who died suddenly in a scenario similar to the one described. Although all documented VT/ventricular fibrillation episodes were terminated by the device, the episode was not terminated because of the ongoing supraventricular tachycardia. Therefore, no further therapies were available and the patient probably died of fast untreated VT. Programming of a long tachycardia detection interval is dangerous in currently available ICDs.
Initiation of Atrial Fibrillation by Ectopic Beats Originating from the Ostium of the Inferior Vena Cava
Authors: MANSOUR MOUSSA , RUSKIN JEREMY , KEANE DAVID
Published: August 2003

Initiation of AF from IVC. Ectopic beats triggering atrial fibrillation may arise from atrial myocardial extensions in the pulmonary veins, superior vena cava, coronary sinus, and vein of Marshall. We report two patients with paroxysmal atrial fibrillation initiated by ectopic beats originating from the inferior vena cava. The foci responsible for the ectopic beats were located and ablated, resulting in elimination of atrial fibrillation. Ectopic beats originating from the os of the inferior vena cava can result in atrial fibrillation. This finding may explain the recurrence of atrial fibrillation in some patients after pulmonary vein isolation.
Ventricular Fibrillation: Ablation of a Trigger?
Authors: SALIBA WALID , KARIM AHMAD ABDUL , TCHOU PATRICK , NATALE ANDREA
Published: August 2003

Ventricular Fibrillation: Ablation of a Trigger? We report the case of a patient with recurrent ventricular fibrillation (VF) and no evidence of structural heart disease. VF was consistently initiated by a relatively early‐coupled premature ventricular contraction with identical morphology on each occasion. Treatment with antiarrhythmic agents failed to suppress the arrhythmia. Electrophysiologic testing showed high‐frequency potential at the earliest activation site, and radiofrequency ablation resulted in abolition of the ventricular ectopy with no further VF recurrence. Suppression of VF arising from focal triggers in patients with frequent ventricular ectopy and normal heart can be achieved with catheter ablation, but further studies are needed to evaluate the prevalence of such a mechanism.
Pulmonary Vein Firing Triggering Atrial Fibrillation After Open Heart Surgery
Authors: SAAD EDUARDO B. , SALIBA WALID I. , MARROUCHE NASSIR F. , NATALE ANDREA
Published: August 2003

PV Firing Triggering AF. We report the case of a 44‐year‐old woman with obstructive hypertrophic cardiomyopathy without history of prior arrhythmias who underwent surgical myectomy. She developed symptomatic postoperative atrial fibrillation (AF) that was refractory to antiarrhythmic therapy and could not be adequately rate controlled. AF always was preceded by short and fast runs of atrial tachycardia. In the electrophysiology laboratory, this arrhythmia appeared to originate from the right superior pulmonary vein and conducted with variable degrees of exit block. Pulmonary vein isolation guided by circular mapping was performed, and no further episodes of either atrial tachycardia or AF were noted. This case highlights the potential role of the pulmonary veins in the pathophysiology of postoperative AF.
Symmetry, structural phase transitions and phase diagram of Langmuir monolayers
Authors: Kaganer V. M. , Indenbom V. L.
Published: August 2003

The Landau theory of phase transitions is developed for transitions between condensed phases of langmuir monolayers. The phase diagram is explained by the coupling between the order parameters describing collective tilt of the molecules and ordering of their backbone (zigzag) planes. if the latter ordering is considered as occurring between hexatic phases, then the predictions do not agree with X-ray data on the structure of phases of either fatty acid or long-chain monolayers on the water surface. However this transition can be explained as translational ordering with doubling of the unit cell realized either by herringbone ordering or by alternating orientations of the heads of the molecules in adjacent rows.
Pattern formation in the splay Freedericks transition of a nematic side-group polysiloxane
Authors: Schwenk Norbert , Spiess Hans Wolfgang
Published: August 2003

Periodic director patterns in the magnetic-field induced splay Freedericks transition of a nematic side-group polysiloxane are reported. For this purpose liquid crystal cells (10 μm-500 μm) are studied by polarization microscopy as well as by deuteron NMR. Through this combinatin, the optically observed spatial dependence of the director can be quantitatively analyzed in terms of director distributions extracted from the NMR lineshape. In the equilibrium state of the Freedericks transition (static Freedericks effect), the director exhibits a one-dimensional periodicity perpendicular to the initial director orientation n0. The dynamics of the Freedericks transition involves a transient two-dimensional director pattern, representing convection rolls in which the nonlinear coupling between director rotation and viscous flow of the nematic (back-flow) leads to a reduction of the viscosity.
Dependence of viscoelastic properties on spacer length and molecular weight for a side-chain liquid crystal polymer in a nematic solvent
Authors: Gu D. , Smith S. R. , Jamieson A. M. , Lee M. , Percec V.
Published: August 2003

The light scattering technique was used to investigate the viscoelastic parapeters characterizing director distortions in miscible mixtures of pentacyanobiphenyl (5CB) with a side-chain liquid crystal polymer (LCP) having different spacer lengths (n=2, 3, 5, 7, 11). To separate the elastic constants from the corresponding viscosities, two approaches were attempted: (a) an AC electric field was applied to homeotropically-aligned nematic monodomains of the mixtures, and the field-dependent scattering intensities and director distortion relaxation rates were measured to obtain the twist viscosity γ1 and elastic constant K22; (b) an electric field was applied to a homogeneously aligned monodomain and the votage-dependent capacitance and the threshold voltage were measured to obtain the dielectric constants and the splay elastic constant. The remaining splay viscosity, bend viscosity and bend elastic constant were subsequently determined by fitting the angular-dependent relaxation rates in three scattering geometries which correspond principally to splay, twist, and bend modes of the director distortions ofr nematic miwtures. The additin of liquid crystal polymers cause significant decreases of the relaxation rates for all three distortion modes of 5CB which are due to small decreases in the elastic constants and large increases in the viscosity coefficients. The molecular weight dependence of the viscosities for n=3 is weak. The largest increase in viscosities is found for the LCP with shortest spacer length n=2. The dependence of viscosity on spacer length disappears when n≥5. The anisotropy in the three viscosity incrementsof the nematic mixtures also becomes smaller when n≥5. Our results indicate that, for side-chain LCPs in a nematic solvent, the backbone configurational anisotropy is enhanced by a strong coupling between the mesogenic group in the side-chain and the chain backbone.
Cell organization in soft media due to active mechanosensing
Authors: Bischofs I. B. , Schwarz U. S.
Published: August 2003

Adhering cells actively probe the mechanical properties of their
environment and use the resulting information to position and orient
themselves. We show that a large body of experimental observations can be
consistently explained from one unifying principle, namely that cells
strengthen contacts and cytoskeleton in the direction of large effective
stiffness. Using linear elasticity theory to model the extracellular
environment, we calculate optimal cell organization for several situations of
interest and find excellent agreement with experiments for fibroblasts, both
on elastic substrates and in collagen gels: cells orient in the direction of
external tensile strain; they orient parallel and normal to free and clamped
surfaces, respectively; and they interact elastically to form strings. Our
method can be applied for rational design of tissue equivalents. Moreover, our
results indicate that the concept of contact guidance has to be reevaluated.
We also suggest that cell–matrix contacts are up-regulated by large
effective stiffness in the environment because, in this way, build-up of force
is more efficient.
Rethinking the evolutionary theory of aging: Transfers, not births, shape senescence in social species
Authors: Lee Ronald D.
Published: August 2003

The classic evolutionary theory of aging explains why mortality rises with age: as individuals grow older, less lifetime fertility remains, so continued survival contributes less to reproductive fitness. However, successful reproduction often involves intergenerational transfers as well as fertility. In the formal theory offered here, age-specific selective pressure on mortality depends on a weighted average of remaining fertility (the classic effect) and remaining intergenerational transfers to be made to others. For species at the optimal quantity–investment tradeoff for offspring, only the transfer effect shapes mortality, explaining postreproductive survival and why juvenile mortality declines with age. It also explains the evolution of lower fertility, longer life, and increased investments in offspring.
Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration
Authors: Oike Yuichi , Yasunaga Kunio , Ito Yasuhiro , Matsumoto Shun-ichiro , Maekawa Hiromitsu , Morisada Tohru , Arai Fumio , Nakagata Naomi , Takeya Motohiro , Masuho Yasuhiko , Suda Toshio
Published: August 2003

We report here the identification of an angiopoietin-related growth factor
(AGF). To examine the biological function of AGF
in vivo
, we created
transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF
mice exhibited swollen and reddish ears, nose and eyelids. Histological
analyses of K14-AGF mice revealed significantly thickened epidermis and a
marked increase in proliferating epidermal cells as well as vascular cells in
the skin compared with nontransgenic controls. In addition, we found rapid
wound closure in the healing process and an unusual closure of holes punched
in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in
platelets and mast cells, and detected at wounded skin, whereas there was no
expression of AGF detected in normal skin tissues, suggesting that AGF derived
from these infiltrated cells affects epidermal proliferation and thereby plays
a role in the wound healing process. These findings demonstrate that
biological functions of AGF in epidermal keratinocytes could lead to novel
therapeutic strategies for wound care and epidermal regenerative medicine.
An interacting network of T-box genes directs gene expression and fate in the zebrafish mesoderm
Authors: Goering Lisa M. , Hoshijima Kazuyuki , Hug Barbara , Bisgrove Brent , Kispert Andreas , Grunwald David Jonah
Published: August 2003

T-box genes encode transcription factors that play critical roles in generating the vertebrate body plan. In many developmental fields, multiple T-box genes are expressed in overlapping domains, establishing broad regions in which different combinations of T-box genes are coexpressed. Here we demonstrate that three T-box genes expressed in the zebrafish mesoderm,no tail, spadetail, andtbx6, operate as a network of interacting genes to regulate region-specific gene expression and developmental fate. Loss-of-function and gain-of-function genetic analyses reveal three kinds of interactions among the T-box genes: combinatorial interactions that generate new regulatory functions, additive contributions to common developmental pathways, and competitive antagonism governing downstream gene expression. We propose that T-box genes, like Hox genes, often function within gene networks comprised of related family members.
A cytokinetic function of
Drosophila
ORC6 protein resides in a domain distinct from its replication activity
Authors: Chesnokov Igor N. , Chesnokova Olga N. , Botchan Michael
Published: August 2003

Coordination between separate pathways may be facilitated by the
requirements for common protein factors, a finding congruent with the link
between proteins regulating DNA replication with other important cellular
processes. We report that the smallest of
Drosophila
origin
recognition complex subunits, Orc6, was found in embryos and cell culture
localized to the cell membrane and cleavage furrow during cell division as
well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with
the
Drosophila
peanut (pnut), a member of the septin family of
proteins important for cell division. This interaction, mediated by a distinct
C-terminal domain of Orc6, was substantiated in
Drosophila
cells by
coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6
expression with double-stranded RNA resulted in a formation of multinucleated
cells and also reduced DNA replication. Deletion of the C-terminal
Orc6–peanut interaction domain and subsequent overexpression of the Orc6
mutant protein resulted in the formation of multinucleated cells that had
replicated DNA. This mutant protein does not localize to the membrane or
cleavage furrows. These results suggest that Orc6 has evolved a domain
critical mainly for cytokinesis.
A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation
Authors: Martin Emil , Sharina Iraida , Kots Alexander , Murad Ferid
Published: August 2003

Heterodimeric αβ soluble guanylyl cyclase (sGC) is a recognized
receptor for nitric oxide (NO) and mediates many of its physiological
functions. Although it has been clear that the heme moiety coordinated by
His-105 of the β subunit is crucial for mediating the activation of the
enzyme by NO, it is not understood whether the heme moiety plays any role in
the function of the enzyme in the absence of NO. Here we analyze the effects
of biochemical and genetic removal of heme and its reconstitution on the
activity of the enzyme. Detergent-induced loss of heme from the wild-type
αβ enzyme resulted in several-fold activation of the enzyme. This
activation was inhibited after hemin reconstitution. A heme-deficient mutant
αβCys-105 with Cys substituted for His-105 was constitutively
active with specific activity approaching the activity of the wild-type enzyme
activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT
treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with
ferrous heme was activated by NO and CO alone and showed additive effects
between gaseous effectors and the allosteric activator
5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1
H
-pyrazolo[3,4-
b
]pyridin-3-yl]-pyrimidin-4-ylamine.
We propose that the heme moiety through its coordination with His-105 of the
β subunit acts as an endogenous inhibitor of sGC. Disruption of the
heme-coordinating bond induced by binding of NO releases the restrictions
imposed by this bond and allows the formation of an optimally organized
catalytic center in the heterodimer.
Regulation of gonadotropin gene expression by Müllerian inhibiting substance
Authors: Bédécarrats Grégoy Y. , O'Neill Francis H. , Norwitz Errol R. , Kaiser Ursula B. , Teixeira Jose
Published: August 2003

In addition to its role in causing Müllerian duct regression,
Müllerian inhibiting substance (MIS) is implicated in the regulation of
steroidogenesis, breast and prostate growth, and ovarian follicle recruitment,
all of which are processes controlled or influenced by the
hypothalamic–pituitary–gonadal axis. Whereas the direct effect of
MIS on gonadal, prostate, and breast cells is under investigation, the ability
of MIS to modulate pituitary function, thereby affecting those tissues
indirectly, has not yet been studied. Using LβT2 cells, a murine
gonadotrope-derived cell line, we have evaluated the effects of MIS on the
expression of the gonadotropin genes. We show that both LβT2 cells and
adult rat pituitaries express MIS type II receptor (MISRII) mRNA. Within 2 h,
follicle-stimulating hormone β subunit (FSHβ) mRNA levels are
significantly induced by addition of MIS to LβT2 cells and remain
elevated through 8 h of treatment. Transcriptional activation of both the
FSHβ and luteinizing hormone β subunit (LHβ) gene promoters was
observed by MIS, which enhances the effect of gonadotropin-releasing hormone
(GnRH) agonist on the FSHβ gene promoter and synergizes with the GnRH
agonist to stimulate LHβ gene promoter activity. Addition of MIS to
LβT2 cells stimulates the activity of the rat LHβ gene promoter with
as little as 1 μg/ml and in a dose-dependent manner. These studies report
both MISRII expression in rat pituitary cells and a gonadotrope-derived cell
line and MIS-mediated activation of LHβ and FSHβ gene expression,
and suggest that MIS may modulate the
hypothalamic–pituitary–gonadal axis at more than one level.
AthPEX10, a nuclear gene essential for peroxisome and storage organelle formation during
Arabidopsis
embryogenesis
Authors: Schumann Uwe , Wanner Gerhard , Veenhuis Marten , Schmid Markus , Gietl Christine
Published: August 2003

In yeasts and mammals,
PEX10
encodes an integral membrane protein
with a C
3
HC
4
RING finger motif in its C-terminal domain
and is required for peroxisome biogenesis and matrix protein import. In
humans, its dysfunction in peroxisome biogenesis leads to severe Zellweger
Syndrome and infantile Refsum disease. Here we show that dysfunction of a
homologous gene in
Arabidopsis
leads to lethality at the heart stage
of embryogenesis, impairing the biogenesis of peroxisomes, lipid bodies, and
protein bodies. In a T-DNA insertion mutant disrupting the fourth exon of the
AthPEX10
gene, ultrastructural analyses fail to detect peroxisomes
characteristic for wild-type embryogenesis. Storage triacyl glycerides are not
assembled into lipid bodies (oil bodies; oleosomes) surrounded by the
phospholipid–protein monolayer membrane. Instead, the dysfunctional
monolayer membranes, which derive from the bilayer membrane of the endoplasmic
reticulum, accumulate in the cytosol. Concomitantly the transfer of the
storage proteins from their site of synthesis at the endoplasmic reticulum to
the vacuoles is disturbed. The mutant can be rescued by transformation with
wild-type
AthPEX10
cDNA. Transformants of wild-type
Hansenula
polymorpha
cells with the
AthPEX10
cDNA did produce the encoded
protein without targeting it to peroxisomes. Additionally, the cDNA could not
complement a
Hansenula pex10
mutant unable to form peroxisomes. The
ultrastructural knockout phenotype of
Ath
PEX10p suggests that this
protein in
Arabidopsis
is essential for peroxisome, oleosome, and
protein transport vesicle formation.
The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization
Authors: Gao Xiang-Dong , Albert Stefan , Tcheperegine Serguei E. , Burd Christopher G. , Gallwitz Dieter , Bi Erfei
Published: August 2003

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80–100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.
Phosphatidylinositol phosphate 5-kinase Iβ recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis
Authors: Padrón David , Wang Ying Jie , Yamamoto Masaya , Yin Helen , Roth Michael G.
Published: August 2003

Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms α, β, or γ in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIβ was inhibited with small interference RNA in HeLa cells, expression of PIP5KIα was also reduced slightly, but PIP5KIγ expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIγ could not compensate for loss of PIP5KIβ. When expression of PIP5KIα was reduced, expression of both PIP5KIβ and PIP5KIγ increased and PIP2 levels did not change. A similar increase of PIP5KIα and PIP5KIβ occurred when PIP5KIγ was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIβ.
The tyrosine phosphatase CD148 is excluded from the immunologic synapse and down-regulates prolonged T cell signaling
Authors: Lin Joseph , Weiss Arthur
Published: August 2003

CD148 is a receptor-like protein tyrosine phosphatase up-regulated on T cells after T cell receptor (TCR) stimulation. To examine the physiologic role of CD148 in TCR signaling, we used an inducible CD148-expressing Jurkat T cell clone. Expression of CD148 inhibits NFAT (nuclear factor of activated T cells) activation induced by soluble anti-TCR antibody, but not by antigen-presenting cells (APCs) loaded with staphylococcal enterotoxin superantigen (SAg) or immobilized anti-TCR antibody. Immunofluorescence microscopy revealed that the extracellular domain of CD148 mediates its exclusion from the immunologic synapse, sequestering it from potential substrates. Targeting of the CD148 phosphatase domain to the immunologic synapse potently inhibited NFAT activation by all means of triggering through the TCR. These data lead us to propose a model where CD148 function is regulated in part by exclusion from substrates in the immunologic synapse. Upon T cell–APC disengagement, CD148 can then access and dephosphorylate substrates to down-regulate prolongation of signaling.
Spatial restriction of α4 integrin phosphorylation regulates lamellipodial stability and α4β1-dependent cell migration
Authors: Goldfinger Lawrence E. , Han Jaewon , Kiosses William B. , Howe Alan K. , Ginsberg Mark H.
Published: August 2003

Întegrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from α4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient α4-mediated migration requires spatial control of α4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated α4 accumulated along the leading edge. Blocking α4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced α4-dependent migration and lamellipodial stability. α4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-α4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated α4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with α4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.
A Role of the Fast ATP-gated P2X1 Cation Channel in Thrombosis of Small Arteries In Vivo
Authors: Hechler Béatrice , Lenain Nadège , Marchese Patrizia , Vial Catherine , Heim Véronique , Freund Monique , Cazenave Jean-Pierre , Cattaneo Marco , Ruggeri Zaverio M. , Evans Richard , Gachet Christian
Published: August 2003

The P2X1 receptor is a fast ATP-gated cation channel expressed in blood platelets, where its role has been difficult to assess due to its rapid desensitization and the lack of pharmacological tools. In this paper, we have used P2X1−/− and wild-type mouse platelets, treated with apyrase to prevent desensitization, to demonstrate the function of P2X1 in the response to thrombogenic stimuli. In vitro, the collagen-induced aggregation and secretion of P2X1-deficient platelets was decreased, as was adhesion and thrombus growth on a collagen-coated surface, particularly when the wall shear rate was elevated. In vivo, the functional role of P2X1 could be demonstrated using two models of platelet-dependent thrombotic occlusion of small arteries, in which blood flow is characterized by a high shear rate. The mortality of P2X1−/− mice in a model of systemic thromboembolism was reduced and the size of mural thrombi formed after a laser-induced vessel wall injury was decreased as compared with normal mice, whereas the time for complete thrombus removal was shortened. Overall, the P2X1 receptor appears to contribute to the formation of platelet thrombi, particularly in arteries in which shear forces are high.
Molecular Motions of the Outer Ring of Charge of the Sodium Channel
Authors: Xiong Wei , Li Ronald A. , Tian Yanli , Tomaselli Gordon F.
Published: August 2003

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain β1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the α carbons of these residues of no more than 7 Å. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.
The Nature of the Controversy over Time-Symmetric Quantum Counterfactuals
Authors: Kastner Ruth E.
Published: August 2003

It is proposed that the recent controversy over “time-symmetric quantum counterfactuals” (TSQCs), based on the Aharonov-Bergmann-Lebowitz Rule for measurements of pre- and post-selected systems, can be clarified by taking TSQCs to be counterfactuals with a specific type of compound antecedent. In that case, inconsistency proofs such as that of Sharp and Shanks (1993) are not applicable, and the main issue becomes not whether such statements are true, but whether they are nontrivial. The latter question is addressed and answered in the negative. Thus it is concluded that TSQCs, understood as counterfactuals with a compound antecedent, are true but only trivially so, and provide no new contingent information about specific quantum systems (except in special cases already identified in literature).
TWISTED DWARF1, a Unique Plasma Membrane-anchored Immunophilin-like Protein, Interacts withArabidopsisMultidrug Resistance-like Transporters AtPGP1 and AtPGP19
Authors: Geisler Markus , Kolukisaoglu H. Üner , Bouchard Rodolphe , Billion Karla , Berger Joachim , Saal Beate , Frangne Nathalie , Koncz-Kálmán Zsuzsanna , Koncz Csaba , Dudler Robert , Blakeslee Joshua J. , Murphy Angus S. , Martinoia Enrico , Schulz Burkhard
Published: August 2003

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrugresistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.
Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding
Authors: Shen Ying H. , Godlewski Jakub , Bronisz Agnieszka , Zhu Jun , Comb Michael J. , Avruch Joseph , Tzivion Guri
Published: August 2003

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.
Modulation of Myoblast Fusion by Caveolin-3 in Dystrophic Skeletal Muscle Cells: Implications for Duchenne Muscular Dystrophy and Limb-Girdle Muscular Dystrophy-1C
Authors: Volonte Daniela , Peoples Aaron J. , Galbiati Ferruccio
Published: August 2003

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-3 is the principal structural component of caveolae in skeletal muscle cells in vivo. We have recently generated caveolin-3 transgenic mice and demonstrated that overexpression of wild-type caveolin-3 in skeletal muscle fibers is sufficient to induce a Duchenne-like muscular dystrophy phenotype. In addition, we have shown that caveolin-3 null mice display mild muscle fiber degeneration and T-tubule system abnormalities. These data are consistent with the mild phenotype observed in Limb-girdle muscular dystrophy-1C (LGMD-1C) in humans, characterized by a ∼95% reduction of caveolin-3 expression. Thus, caveolin-3 transgenic and null mice represent valid mouse models to study Duchenne muscular dystrophy (DMD) and LGMD-1C, respectively, in humans. Here, we derived conditionally immortalized precursor skeletal muscle cells from caveolin-3 transgenic and null mice. We show that overexpression of caveolin-3 inhibits myoblast fusion to multinucleated myotubes and lack of caveolin-3 enhances the fusion process. M-cadherin and microtubules have been proposed to mediate the fusion of myoblasts to myotubes. Interestingly, we show that M-cadherin is downregulated in caveolin-3 transgenic cells and upregulated in caveolin-3 null cells. For the first time, variations of M-cadherin expression have been linked to a muscular dystrophy phenotype. In addition, we demonstrate that microtubules are disorganized in caveolin-3 null myotubes, indicating the importance of the cytoskeleton network in mediating the phenotype observed in these cells. Taken together, these results propose caveolin-3 as a key player in myoblast fusion and suggest that defects of the fusion process may represent additional molecular mechanisms underlying the pathogenesis of DMD and LGMD-1C in humans.
Loss of SEC-23 inCaenorhabditis elegansCauses Defects in Oogenesis, Morphogenesis, and Extracellular Matrix Secretion
Authors: Roberts Brett , Clucas Caroline , Johnstone Iain L.
Published: August 2003

SEC-23 is a component of coat protein complex II (COPII)-coated vesicles involved in the endoplasmic reticulum-to-Golgi transport pathway of eukaryotes. During postembryonic life, Caenorhabditis elegans is surrounded by a collagenous exoskeleton termed the cuticle. From a screen for mutants defective in cuticle secretion, we identified and characterized a sec-23 mutant of C. elegans. By sequence homology, C. elegans has only the single sec-23 gene described herein. In addition to the cuticle secretion defect, mutants fail to complete embryonic morphogenesis. However, they progress through the earlier stages of embryogenesis, including gastrulation, and achieve substantial morphogenesis before death. We demonstrated a maternal component of SEC-23 function sufficient for progression through the earlier stages of embryogenesis and explaining the limited phenotype of the zygotic mutant. By RNA-mediated interference, we investigated the effects of perturbing COPII function during various postembryonic stages. During larval stages, major defects in cuticle synthesis and molting were observed. In the adult hermaphrodite, reduction of SEC-23 function by RNA-mediated interference caused a rapid onset of sterility, with defects in oogenesis including early maturation of the germline nuclei, probably a result of the observed loss of the GLP-1 receptor from the membrane surfaces adjacent to the developing germline nuclei.
PIKfyve Controls Fluid Phase Endocytosis but Not Recycling/Degradation of Endocytosed Receptors or Sorting of Procathepsin D by Regulating Multivesicular Body Morphogenesis
Authors: Ikonomov Ognian C. , Sbrissa Diego , Foti Michelangelo , Carpentier Jean-Louis , Shisheva Assia
Published: August 2003

The mammalian phosphatidylinositol (PtdIns) 5-P/PtdIns 3,5-P2–producing kinase PIKfyve has been implicated in maintaining endomembrane homeostasis in mammalian cells. To address the role of PIKfyve in trafficking processes, we examined the functioning of the biosynthetic, endocytic, and recycling pathways in stable human embryonic kidney 293 cell lines inducibly expressing the wild-type or kinase-defective dominant-negative form. PIKfyveWTor PIKfyveK1831Eexpression did not affect the processing and lysosomal targeting of newly synthesized procathepsin D. Likewise the rates of transferrin uptake/recycling or epidermal growth factor receptor degradation were not altered upon expression of either protein. In contrast, PIKfyveK1831Ebut not PIKfyveWTexpression markedly impaired the late uptake of fluid phase marker horseradish peroxidase. Inspection of the organelle morphology by confocal microscopy with specific markers in COS cells transiently expressing PIKfyveK1831Eshowed the Golgi apparatus, end lysosomes, and the recycling compartment indistinguishable from nontransfected cells, despite the dramatic PIKfyveK1831E-induced endomembrane vacuolation. In contrast, we observed a striking effect on the late endocytic compartment, marked by disruption of the dextran-labeled perinuclear endosomal compartment and formation of dispersed enlarged vesicles. Electron microscopy identified the cytoplasmic vacuoles in the PIKfyveK1831E-expressing human embryonic kidney 293 cells as enlarged multivesicular body-like structures with substantially lower number of internal vesicles and membrane whorls. Together, these data indicate that PIKfyve selectively regulates the sorting and traffic of peripheral endosomes containing lysosomaly directed fluid phase cargo through controlling the morphogenesis and function of multivesicular bodies.
Importin β Negatively Regulates Nuclear Membrane Fusion and Nuclear Pore Complex Assembly
Authors: Harel Amnon , Chan Rene C. , Lachish-Zalait Aurelie , Zimmerman Ella , Elbaum Michael , Forbes Douglass J.
Published: August 2003

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin β negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin β is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin β down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin β to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin β 45–462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin β-regulation of NPC assembly. Excess full-length importin β and β 45–462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin β NPC block, which maps downstream of GTPγS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 μM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin β. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.
Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice
Authors: Houghtaling Scott , Timmers Cynthia , Noll Meenakshi , Finegold Milton J. , Jones Stephen N. , Meyn M. Stephen , Grompe Markus
Published: August 2003

Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA damage, bone marrow failure, congenital defects, and cancer. To further investigate the in vivo function of the FA pathway, mice with a targeted deletion in the distally acting FA geneFancd2were created. Similar to human FA patients and other FA mouse models,Fancd2mutant mice exhibited cellular sensitivity to DNA interstrand cross-links and germ cell loss. In addition, chromosome mispairing was seen in male meiosis. However,Fancd2mutant mice also displayed phenotypes not observed in other mice with disruptions of proximal FA genes. These include microphthalmia, perinatal lethality, and epithelial cancers, similar to mice withBrca2/Fancd1hypomorphic mutations. These additional phenotypes were not caused by defects in the ATM-mediated S-phase checkpoint, which was intact in primaryFancd2mutant fibroblasts. The phenotypic overlap betweenFancd2-null andBrca2/Fancd1hypomorphic mice is consistent with a common function for both proteins in the same pathway, regulating genomic stability.