bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel paracrine signaling mechanism in the ocular ciliary epithelium
Authors: Hirata Keiji , Nathanson Michael H. , Sears Marvin L.
Published: July 2002

The ciliary body contains an epithelial bilayer consisting of an outer pigmented cell layer (PE) and an inner nonpigmented cell layer (NPE) responsible for aqueous humor secretion. Secretion may be mediated in part by cytosolic Ca
2+
concentration ([Ca
2+
]
i
), but whether or how the two layers could coordinate their Ca
2+
signals to regulate secretion is unclear. To investigate interactions between PE and NPE, we examined [Ca
2+
]
i
signaling in isolated intact ciliary epithelial bilayers using confocal microscopy. Phenylephrine selectively increased [Ca
2+
]
i
in PE and acetylcholine increased [Ca
2+
]
i
in NPE, but epinephrine increased [Ca
2+
]
i
in both layers. This increase spread from PE to NPE, and [Ca
2+
]
i
signaling across the bilayer remained coordinated during [Ca
2+
]
i
oscillations. All epinephrine-induced [Ca
2+
]
i
signaling was blocked by the α
1
-adrenergic antagonist prazosin, whereas signaling in the NPE but not PE was blocked by the β-adrenergic antagonist propranolol, the gap junction blockers octanol and 18α-glycyrrhetinic acid, or the A kinase inhibitor
R
p
diastereomer of adenosine 3′,5′-cyclic monophosphothioate. The β-adrenergic agonist isoproterenol failed to increase Ca
2+
by itself, but isoproterenol plus phenylephrine-induced [Ca
2+
]
i
signals across the bilayer similar to those induced by epinephrine. Finally, isoproterenol increased cell-to-cell spread of lucifer yellow via gap junctions, whereas cell-to-cell spread of [Ca
2+
]
i
signals could be induced by photorelease of caged inositol 1,4,5-trisphosphate. Thus, calcium signals are coordinated in the epithelial bilayer so that adrenergic stimulation can increase [Ca
2+
]
i
in NPE, but only if NPE are primed by activation of endogenous adenylyl cyclase, whereupon they receive stimulation from adjacent PE via gap junctions. This novel interplay between endocrine and paracrine pathways may coordinate [Ca
2+
]
i
signaling across the ciliary epithelial bilayer.
A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation
Authors: Murray L. E. , Rowley N. , Dawes I. W. , Johnston G. C. , Singer R. A.
Published: July 2002

Dimorphic growth of the budding yeast
Saccharomyces cerevisiae
is regulated by the quality of the nitrogen supply. On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells. Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5′-CUG anticodon (tRNA
CUG
). Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source. The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene
CAR1
and undergo precocious sporulation in nitrogen-rich media. Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development.
unc
-
1
: A stomatin homologue controls sensitivity to volatile anesthetics in
Caenorhabditis elegans
Authors: Rajaram S. , Sedensky M. M. , Morgan P. G.
Published: July 2002

To identify sites of action of volatile anesthetics, we are studying genes in a functional pathway that controls sensitivity to volatile anesthetics in the nematode
Caenorhabditis elegans
. The
unc-1
gene occupies a central position in this pathway. Different alleles of
unc-1
have unique effects on sensitivity to the different volatile anesthetics. UNC-1 shows extensive homology to human stomatin, an integral membrane protein thought to regulate an associated ion channel. We postulate that UNC-1 has a direct effect on anesthetic sensitivity in
C. elegans
and may represent a molecular target for volatile anesthetics.
Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores
Authors: Csutora Peter , Su Zhengchang , Kim Hak Yong , Bugrim Andrej , Cunningham Kyle W. , Nuccitelli Richard , Keizer Joel E. , Hanley Michael R. , Blalock J. Edwin , Marchase Richard B.
Published: July 2002

Depletion of endoplasmic reticulum Ca
2+
stores leads to the entry of extracellular Ca
2+
into the cytoplasm, a process termed capacitative or store-operated Ca
2+
entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca
2+
stores were depleted by chemical and genetic means, respectively. After microinjection into
Xenopus laevis
oocytes, the extracts elicited a wave of increased cytoplasmic free Ca
2+
([Ca
2+
]
i
) that spread from the point of injection across the oocyte. Extracts from cells with replete organellar Ca
2+
stores were inactive. The increases depended on extracellular Ca
2+
, were unaffected by the inositol 1,4,5-trisphosphate (IP
3
) inhibitor heparin or an anti-IP
3
receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation. Confocal microscopy revealed that [Ca
2+
]
i
increases were most pronounced at the periphery of the oocyte. The patterns of [Ca
2+
]
i
increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca
2+
influx. In addition, I
CRAC
, a
C
a
2+
r
elease-
a
ctivated
C
a
2+
current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca
2+
chelator or IP
3
. These data support the existence in yeast and mammalian cells depleted of Ca
2+
stores of a functionally conserved diffusible calcium influx factor that directly activates Ca
2+
influx.
The transition to agricultural sustainability
Authors: Ruttan Vernon W.
Published: July 2002

The transition to sustainable growth in agricultural production
during the 21st century will take place within the context of a
transition to a stable population and a possible transition to a stable
level of material consumption. If the world fails to successfully
navigate a transition to sustainable growth in agricultural production,
the failure will be due more to a failure in the area of institutional
innovation than to resource and environmental constraints.
Selectable viruses and altered susceptibility mutants in
Arabidopsis thaliana
Authors: Whitham Steven A. , Yamamoto Miki L. , Carrington James C.
Published: July 2002

The genetic basis for susceptibility or nonsusceptibility of plants to viruses is understood poorly. Two selectable tobacco etch virus (TEV) strains were developed for identification of
Arabidopsis thaliana
mutants with either gain-of-susceptibility or loss-of-susceptibility phenotypes. These strains conferred a conditional-survival phenotype to
Arabidopsis
based on systemic expression of herbicide resistance or proherbicide sensitivity genes, thereby facilitating mass selections and screens for
Arabidopsis
mutants that enhance or suppress TEV replication, cell-to-cell movement, or long-distance movement. A multicomponent mechanism that restricts systemic invasion of TEV was identified through isolation of gain-of-susceptibility mutants with alterations at two loci.
The neurological basis of conscious color perception in a blind patient
Authors: Zeki S. , Aglioti S. , McKeefry D. , Berlucchi G.
Published: July 2002

We have studied patient PB, who, after an electric shock that led to vascular insufficiency, became virtually blind, although he retained a capacity to see colors consciously. For our psychophysical studies, we used a simplified version of the Land experiments [Land, E. (1974)
Proc. R. Inst. G. B.
47, 23–58] to learn whether color constancy mechanisms are intact in him, which amounts to learning whether he can assign a constant color to a surface in spite of changes in the precise wavelength composition of the light reflected from that surface. We supplemented our psychophysical studies with imaging ones, using functional magnetic resonance, to learn something about the location of areas that are active in his brain when he perceives colors. The psychophysical results suggested that color constancy mechanisms are severely defective in PB and that his color vision is wavelength-based. The imaging results showed that, when he viewed and recognized colors, significant increases in activity were restricted mainly to V1-V2. We conclude that a partly defective color system operating on its own in a severely damaged brain is able to mediate a conscious experience of color in the virtually total absence of other visual abilities.
Coordinated regulation of fetal and maternal prostaglandins directs successful birth and postnatal adaptation in the mouse
Authors: Reese Jeff , Paria Bibhash C. , Brown Naoko , Zhao Xuemei , Morrow Jason D. , Dey Sudhansu K.
Published: July 2002

Cyclooxygenase (COX)-derived prostaglandins (PGs) regulate numerous maternal–fetal interactions during pregnancy. PGs stimulate uterine contractions and prepare the cervix for parturition, whereas in the fetus, PGs maintain patency of the ductus arteriosus (DA), a vascular shunt that transmits oxygenated placental blood to the fetal systemic circulation. However, the origin and site of action of these PGs remain undefined. To address this, we analyzed mice lacking COX-1 (null mutation) or COX-2 (pharmacologic inhibition) or pups with a double null mutation. Our results show that COX-1 in the uterine epithelium is the major source of PGs during labor and that COX-1−/−females experience parturition failure that is reversible by exogenous PGs. Using embryo transfer experiments, we also show that successful delivery occurs in COX-1−/−recipient mothers carrying wild-type pups, establishing the sufficiency of fetal PGs for parturition. Although patency of the DA is PG dependent, neither COX-1 nor COX-2 expression was detected in the fetal or postnatal DA, and offspring with a double null mutation died shortly after birth with open DAs. These results suggest that DA patency depends on circulating PGs acting on specific PG receptors within the DA. Collectively, these findings demonstrate the coordinated regulation of fetal and maternal PGs at the time of birth but raise concern regarding the use of selective COX inhibitors for the management of preterm labor.
Position-dependent linkages of fibronectin– integrin–cytoskeleton
Authors: Nishizaka Takayuki , Shi Qing , Sheetz Michael P.
Published: July 2002

Position-dependent cycling of integrin interactions with both the cytoskeleton and extracellular matrix (ECM) is essential for cell spreading, migration, and wound healing. Whether there are regional changes in integrin concentration, ligand affinity or cytoskeleton crosslinking of liganded integrins has been unclear. Here, we directly demonstrate a position-dependent binding and release cycle of fibronectin–integrin–cytoskeleton interactions with preferential binding at the front of motile 3T3 fibroblasts and release at the endoplasm–ectoplasm boundary. Polystyrene beads coated with low concentrations of an integrin-binding fragment of fibronectin (fibronectin type III domains 7–10) were 3–4 times more likely to bind to integrins when placed within 0.5 microns vs. 0.5–3 microns from the leading edge. Integrins were not concentrated at the leading edge, nor did anti-integrin antibody-coated beads bind preferentially at the leading edge. However, diffusing liganded integrins attached to the cytoskeleton preferentially at the leading edge. Cytochalasin inhibited edge binding, which suggested that cytoskeleton binding to the integrins could alter the avidity for ligand beads. Further, at the ectoplasm–endoplasm boundary, the velocity of bead movement decreased, diffusive motion increased, and approximately one-third of the beads were released into the medium. We suggest that cytoskeleton linkage of liganded integrins stabilizes integrin-ECM bonds at the front whereas release of cytoskeleton-integrin links weakens integrin-ECM bonds at the back of lamellipodia.
Molecular genetics of pattern formation in the inner ear: Do compartment boundaries play a role?
Authors: Brigande John V. , Kiernan Amy E. , Gao Xiaoying , Iten Laurie E. , Fekete Donna M.
Published: July 2002

The membranous labyrinth of the inner ear establishes a precise
geometrical topology so that it may subserve the functions of hearing
and balance. How this geometry arises from a simple ectodermal placode
is under active investigation. The placode invaginates to form the otic
cup, which deepens before pinching off to form the otic vesicle. By the
vesicle stage many genes expressed in the developing ear have assumed
broad, asymmetrical expression domains. We have been exploring the
possibility that these domains may reflect developmental compartments
that are instrumental in specifying the location and identity of
different parts of the ear. The boundaries between compartments are
proposed to be the site of inductive interactions required for this
specification. Our work has shown that sensory organs and the
endolymphatic duct each arise near the boundaries of broader gene
expression domains, lending support to this idea. A further prediction
of the model, that the compartment boundaries will also represent
lineage-restriction compartments, is supported in part by fate mapping
the otic cup. Our data suggest that two lineage-restriction boundaries
intersect at the dorsal pole of the otocyst, a convergence that may be
critical for the specification of endolymphatic duct outgrowth. We
speculate that the patterning information necessary to establish these
two orthogonal boundaries may emanate, in part, from the hindbrain. The
compartment boundary model of ear development now needs to be tested
through a variety of experimental perturbations, such as the removal of
boundaries, the generation of ectopic boundaries, and/or changes
in compartment identity.
Explaining Post-Soviet Ethnic Conflicts: Using Regime Type to Discern the Impact and Relative Importance of Objective Antecedents
Authors: Horowitz Shale
Published: July 2002

The collapse of the Soviet Union unleashed a number of violent, usually secessionist ethnic conflicts. These conflicts were typically intensified (or “escalated”) by foreign intervention. Although there is a great deal of consensus about the fundamental forces driving these conflicts and their escalation, there remain considerable theoretical differences about how to understand these factors and assess their relative importance. These differences mirror debates in the broader literature on national identity and its consequences. This article seeks to clarify these debates by elucidating some theoretical distinctions among the factors taken to contribute to the outbreak and escalation of violent ethnic conflicts. These distinctions are then applied to post-Soviet conflicts in Azerbaijan, Georgia, Moldova, and Tajikistan. The case studies bear out the relevance of the theoretical distinctions, but also reveal the difficulty of applying them unambiguously in any given case. These examples form the basis for some concluding suggestions. These suggestions aim to maximize the clarity with which theoretically distinct causal factors are empirically distinguished for purposes of testing hypotheses.
Distribution of Inorganic Mercury in Liver and Kidney of Beluga and Bowhead Whales Through Autometallographic Development of Light Microscopic Tissue Sections
Authors: Woshner Victoria M. , O'Hara Todd M. , Eurell Jo Ann , Wallig Matthew A. , Bratton Gerald R. , Suydam Robert S. , Beasley Val R.
Published: July 2002

Inorganic mercury was localized through autometallography (AMG) in kidney and liver of free-ranging, subsistence-harvested beluga ( Delphinapterus leucas; n = 20) and bowhead ( Balaena mysticetus ; n = 5) whales. AMG granules were not evident in bowhead tissues, confirming nominal mercury (Hg) concentrations (range = 0.011 to 0.038 ug/g ww for total Hg). In belugas, total Hg ranged from 0.30 to 17.11 and from 0.33 to 82.47 ug/g ww in liver and kidney, respectively. AMG granules were restricted to cortical tubular epithelial cytoplasm in belugas with lower tissue burdens ; whales with higher tissue burdens had granules throughout the uriniferous tubular epithelium. In liver, AMG granular densities differed between lobular zones, concentrating in stellate macrophages and bile cannalicular domains of hepatocytes. AMG granules aggregated in periportal regions in belugas with lower hepatic Hg concentrations, yet among whales with higher Hg, AMG granule deposition extended to pericentral and midzonal regions of liver lobules. Mean areas occupied by AMG granules correlated well with hepatic Hg concentrations and age. In beluga livers, AMG staining density was not associated with lipofuscin quantity (an index of oxidative damage). Occasionally, AMG granules and lipofuscin were colocalized, but more often were not, implying that Hg was not a prominent factor in hepatic lipofuscin deposition in belugas.
Pregnancy Dating in the Rat: Placental Morphology and Maternal Blood Parameters
Authors: De Rijk Eveline P.C.T. , Van Esch Eric , Flik Gert
Published: July 2002

The rat is commonly used as a model in studies on embryology and reproduction toxicology. Surprisingly, the current literature does not provide a comprehensive reference data set on placental development in rat. Therefore, we have evaluated morphological changes of the placenta and maternal blood parameters during pregnancy of the Sprague—Dawley rat. The morphologic data presented in this study may be useful as reference material. This study revealed that placental development in the rat is a well-defi ned process, characterized by key synchronized morphological events at specifi c points in time, convenient for laboratory practice and provides the toxicologist with a sensitive tool to distinguish between normal and abnormal placental development and to detect fetal and placental mismatches. During rat pregnancy, signifi cant changes were observed in maternal blood parameters strongly reminiscent of those observed in pregnant women. These changes included: (a) decreased blood cell volume as a result of hemodilution, (b) increased white blood cell counts refl ecting the response of the mother to the fetal allograft, (c) increased blood clotting values, (d) decreased plasma glucose and increased lipid content maximizing fetal glucose availability and maternal energy conservation, and (e) decreased electrolyte values refl ecting plasma volume expansion. It was concluded that the combined data set on placental morphology and maternal blood parameters in pregnant rats provides powerful tools for recognition of abnormal pregnancies.
Drinking goal selection and treatment outcome in out‐patients with mild‐moderate alcohol dependence
Authors: ADAMSON SIMON J. , SELLMAN J. DOUGLAS
Published: July 2002

Selection of drinking goal is examined at baseline, post‐treatment and at 6 months follow‐up for a sample of mild‐moderate alcohol‐dependent out‐patients. Drinking goal is identified as abstinent or controlled drinking, with the latter group being asked to further specify per session and per week drinking limits. Group comparisons for drinking goal post‐treatment show those who were not assigned motivational enhancement therapy, had more drinking days and lower scores on the Alcohol Problems Questionnaire and Internal Motivation were more likely to choose controlled drinking. The only variable to predict independently whether or not the controlled drinking goal was within the promoted drinking guidelines was age, with younger participants more likely to choose a goal above this limit. Goal selection was significantly related to drinking outcome, with those aiming to drink within guidelines having better outcome than those aiming for higher limits. There was no significant difference in drinking outcome category when those aiming for within limits were compared to those aiming for abstinence. Clinical implications of these findings are discussed.
Addiction, Community and State: A Review
Authors: Westermeyer Joseph
Published: July 2002

As clinicians, we are well aware of the importance of drug supply, price, availability, and access in propagating and maintaining illicit drug use, abuse, and addiction. However, we tend to be silent on these topics, leaving them to elected and appointed government leaders. The author proposes that our remaining silent is analogous to an infectious disease expert remaining silent on water supply and sewage in the midst of a cholera epidemic. It is not only unseemly for us to remain silent, it is unprofessional at best and unethical at worst.

Governmental means of addressing the social genesis of addiction and illicit drug supply falls under the general rubric of statecraft. Our elected leaders, our appointed governmental leaders, and our publicly supported workers (e.g., police, courts, schools, health care workers, social agencies) can and do exert influences either favoring or counteracting illicit drug supply, use, abuse, and addiction. Public officials can and do learn the exercise of statecraft in addressing a large range of social needs, such as housing, transportation, security, a safe food supply, and so forth. They can and should develop knowledge, skills, and experience in the exercise of statecraft vis‐a‐vis drugs.

This article reviews the relevant literature on this topic. In addition, the author reflects on his observations on “addiction statecraft” in several countries over the past three decades. He underscores the need to consider elements such as community deterioration, corruption of police and other officials, and social antidrug strategies that have worked as well as those that have not worked. Public leaders must also take stands regarding intervention and treatment programs that are not widely popular, such as those involving methadone. The author argues that we speak out authoritatively on “addiction statecraft.” In undertaking such social roles, we must take care to speak out of our own collective wisdom and experience, rather than out of personal bias or personal agendas.
Two online surveys of Italian Internet audiences: a summary of findings
Authors: Magistretti Stefano
Published: July 2002

This article describes the main results of separate online surveys on two partly differentiated Italian Internet audiences. It argues that the two show more traits in common than dissimilarities and that their common characteristics are widely shared by the millions of Italian Internet users. On a comparison of the sample to the country as a whole, it emerges as better educated, more open-minded, more affluent, and young to middle-aged. These Internet audiences do not trust television, they rely on the Internet to provide them with news, entertainment, goods and services, and they are quite similar to other Internet users elsewhere in western Europe.
Imagining the Web: the social construction of the Internet in Italy
Authors: Pasquali Francesca
Published: July 2002

The article analyses social discourses about the Internet in Italy from the mid-1990s onwards, taking its examples from advertising. Beyond the individual campaigns and their subjects there have been two distinct trends in Internet advertising. The first has made an effort to build the Internet as a cognitive object, the second has presented the Internet as a ‘possible world'. The article aims to account for the ways in which the Web has been thematized in Italy: its fields of reference, and how its possible social and personal uses have been anticipated.
Variability in comprehension strategy use in children with SLI: A dynamical systems account
Authors: Evans Julia L.
Published: July 2002

The aim was to investigate the stability of comprehension strategy use in children with specific language impairment (SLI). According to principles of Dynamic Systems Theory, behaviour is both unstable and more easily affected by changes in external variables during developmental transitions. The study examined this prediction directly by comparing the comprehension strategy use of children with SLI in two different conditions. The subjects included 16 school‐aged children with SLI (ages 6;8−8;5), and eight chronologically age‐matched (CA) controls. One group of children with SLI (n =8) was in a development transition between animacy and word‐order comprehension strategy use, demonstrating the emerging use of word‐order sentence comprehension strategies (SLI‐T). The second group of children with SLI (n =8) was not in a developmental transition (SLI‐N), demonstrating the use of only animacy comprehension strategies characteristic of younger typically developing children. Children were asked to determine the agent in 54 sentences of the forms (NVN, NNV, VNN) with animacy of the noun as a second factor in each of two different conditions designed to vary external processing demands. Analysis of variance and maximum likelihood estimate analysis revealed that comprehension strategies for both groups of children with SLI differed qualitatively from CA controls. In particular, the findings revealed that comprehension strategy use in children with SLI was highly vulnerable to increases in external processing demands as compared with CA controls, suggesting that comprehension strategies in children with SLI are weakly represented regardless of the strategy they use. Findings from the study also illustrate the impact of intrinsic as well as extrinsic contextual factors on the stability and instability of language processing performance in children with SLI.
The Vertebrate GLFG Nucleoporin, Nup98, Is an Essential Component of Multiple RNA Export Pathways
Authors: Powers Maureen A. , Forbes Douglass J. , Dahlberg James E. , Lund Elsebet
Published: July 2002

The 97-kD O-linked glycoprotein, Nup98, is a component of the Xenopus laevis nuclear pore complex and the only vertebrate GLFG nucleoporin identified (Powers, M.A., C. Macauley, F. Masiarz, and D.J. Forbes. 1995. J. Cell Biol. 128:721–736). We have investigated possible roles of xNup98 in the nucleocytoplasmic transport of proteins and RNAs by analyzing the consequences of injecting monospecific polyclonal antibodies to xNup98 into X. laevis oocytes. We show here that nuclear injection of anti-xNup98 inhibited the export of multiple classes of RNAs, including snRNAs, 5S RNA, large ribosomal RNAs, and mRNA. In contrast, the export of tRNA was unaffected. Injection of antixNup98 into the oocyte cytoplasm had no effect on export of any of the RNAs. Significantly, nuclear injection of anti-xNup98 antibodies did not inhibit import of either karyophilic proteins or snRNPs. This latter result is in agreement with our previous finding that Nup98 is not an essential element of the protein import pathway. Thus, Nup98 plays a role specifically in RNA export from the nucleus, and it appears to be an essential component of multiple RNA export pathways.
Rapid Retrograde Tyrosine Phosphorylation of trkA and Other Proteins in Rat Sympathetic Neurons in Compartmented Cultures
Authors: Senger Donna L. , Campenot Robert B.
Published: July 2002

According to the current theory of retrograde signaling, NGF binds to receptors on the axon terminals and is internalized by receptor-mediated endocytosis. Vesicles with NGF in their lumina, activating receptors in their membranes, travel to the cell bodies and initiate signaling cascades that reach the nucleus. This theory predicts that the retrograde appearance of activated signaling molecules in the cell bodies should coincide with the retrograde appearance of the NGF that initiated the signals. However, we observed that NGF applied locally to distal axons of rat sympathetic neurons in compartmented cultures produced increased tyrosine phosphorylation of trkA in cell bodies/ proximal axons within 1 min. Other proximal proteins, including several apparently localized in cell bodies, displayed increased tyrosine phosphorylation within 5–15 min. However, no detectable 125I-NGF appeared in the cell bodies/proximal axons within 30–60 min of its addition to distal axons. Even if a small, undetectable fraction of transported 125I-NGF was internalized and loaded onto the retrograde transport system immediately after NGF application, at least 3–6 min would be required for the NGF that binds to receptors on distal axons just outside the barrier to be transported to the proximal axons just inside the barrier. Moreover, it is unlikely that the tiny fraction of distal axon trk receptors located near the barrier alone could produce a measurable retrograde trk phosphorylation even if enough time was allowed for internalization and transport of these receptors. Thus, our results provide strong evidence that NGF-induced retrograde signals precede the arrival of endocytotic vesicles containing the NGF that induced them. We further suggest that at least some components of the retrograde signal are carried by a propagation mechanism.
Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice
Authors: Carmeliet Peter , Moons Lieve , Dewerchin Mieke , Rosenberg Steven , Herbert Jean-Marc , Lupu Florea , Collen Désiré
Published: July 2002

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of 125I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR−/− and u-PAR+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR+/+ cells, whereas it was present in the pericellular space around u-PAR−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.
A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum
Authors: Abrami Laurence , Fivaz Marc , Glauser Pierre-Etienne , Parton Robert G. , van der Goot F.
Published: July 2002

In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid “rafts.” We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.
Golgi Alkalinization by the Papillomavirus E5 Oncoprotein
Authors: Schapiro Florencia , Sparkowski Jason , Adduci Alex , Suprynowicz Frank , Schlegel Richard , Grinstein Sergio
Published: July 2002

The E5 oncoprotein of bovine papillomavirus type I is a small, hydrophobic polypeptide localized predominantly in the Golgi complex. E5-mediated transformation is often associated with activation of the PDGF receptor (PDGF-R). However, some E5 mutants fail to induce PDGF-R phosphorylation yet retain transforming activity, suggesting an additional mechanism of action. Since E5 also interacts with the 16-kD pore-forming subunit of the vacuolar H+-ATPase (V-ATPase), the oncoprotein could conceivably interfere with the pH homeostasis of the Golgi complex. A pH-sensitive, fluorescent bacterial toxin was used to label this organelle and Golgi pH (pHG) was measured by ratio imaging. Whereas pHG of untreated cells was acidic (6.5), no acidification was detected in E5-transfected cells (pH ∼7.0). The Golgi buffering power and the rate of H+ leakage were found to be comparable in control and transfected cells. Instead, the E5-induced pH differential was attributed to impairment of V-ATPase activity, even though the amount of ATPase present in the Golgi complex was unaltered. Mutations that abolished binding of E5 to the 16-kD subunit or that targeted the oncoprotein to the endoplasmic reticulum abrogated Golgi alkalinization and cellular transformation. Moreover, transformation-competent E5 mutants that were defective for PDGF-R activation alkalinized the Golgi lumen. Neither transformation by sis nor src, two oncoproteins in the PDGF-R signaling pathway, affected pHG. We conclude that alkalinization of the Golgi complex represents a new biological activity of the E5 oncoprotein that correlates with cellular transformation.
P21 and Retinoblastoma Protein Control the Absence of DNA Replication in Terminally Differentiated Muscle Cells
Authors: Mal Asoke , Chattopadhyay Debasis , Ghosh Mrinal K. , Poon Randy Y.C. , Hunter Tony , Harter Marian L.
Published: July 2002

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E–cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state.
Diablo Promotes Apoptosis by Removing Miha/Xiap from Processed Caspase 9
Authors: Ekert Paul G. , Silke John , Hawkins Christine J. , Verhagen Anne M. , Vaux David L.
Published: July 2002

MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.
Involvement of Egr-1/RelA Synergy in Distinguishing T Cell Activation from Tumor Necrosis Factor-α–induced NF-κB1 Transcription
Authors: Cogswell Patricia C. , Mayo Marty W. , Baldwin Albert S.
Published: July 2002

NF-κB is an important transcription factor required for T cell proliferation and other immunological functions. The NF-κB1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-κB. Previously, we and others have demonstrated that NF-κB regulates the NF-κB1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-κB1 encoding transcripts than cells stimulated with tumor necrosis factor-α, despite the fact that both stimuli activate NF-κB. Characterization of the NF-κB1 promoter identified an Egr-1 site which was found to be essential for both the PMA/ PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-κB and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-κB1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-κB1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-κB may have important ramifications in T cell development by upregulating NF-κB1 gene expression.
In Vivo Function of an Interleukin 2 Receptor β Chain (IL-2Rβ)/IL-4Rα Cytokine Receptor Chimera Potentiates Allergic Airway Disease
Authors: Youn Jeehee , Chen Jin , Goenka Shreevrat , Aronica Mark A. , Mora Ana L. , Correa Victor , Sheller James R. , Boothby Mark
Published: July 2002

Strength of T cell receptor (TCR) signaling, coreceptors, costimulation, antigen-presenting cell type, and cytokines all play crucial roles in determining the efficiency with which type 2 T lymphocytes (Th2, Tc2) develop from uncommitted precursors. To investigate in vivo regulatory mechanisms that control the population of type 2 T cells and disease susceptibility, we have created lines of transgenic mice in which expression of a chimeric cytokine receptor (the mouse interleukin 2 receptor β chain [IL-2Rβ] extracellular domain fused to the cytoplasmic tail of IL-4Rα) is targeted to the T lymphoid lineage using the proximal lck promoter. This chimera transduced IL-4–specific signals in response to IL-2 binding and dramatically enhanced type 2 responses (IL-4, IL-5, and immunoglobulin E production) upon in vitro TCR stimulation or in vivo antigen challenge. Thus, type 2 effector function was augmented by IL-4 signals transduced through a chimeric receptor expressed in a T cell–specific manner. This influence was sufficient for establishment of antigen-induced allergic airway hyperresponsiveness on a disease-resistant background (C57BL/6).
A Novel H+ Conductance in Eosinophils
Authors: Bánfi Botond , Schrenzel Jacques , Nüsse Oliver , Lew Daniel P. , Ligeti Erzsébet , Krause Karl-Heinz , Demaurex Nicolas
Published: July 2002

Efficient mechanisms of H+ ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase—in analogy with mitochondrial cytochromes—has an inherent H+ channel activity remains uncertain: electrophysiological studies did not find altered H+ currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H+ currents in human eosinophils. The “classical” H+ current had properties similar to previously described H+ conductances and was present in CGD cells. In contrast, the “novel” type of H+ current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H+ currents; and (e) it was ∼20-fold more sensitive to Zn2+ and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H+ conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H+ extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.
Tumor Rejection by Disturbing Tumor Stroma Cell Interactions
Authors: Ibe Sabrina , Qin Zhihai , Schüler Thomas , Preiss Susanne , Blankenstein Thomas
Published: July 2002

The stroma of solid tumors is a complex network of different cell types. We analyzed stroma cell interactions in two tumor models during cyclophosphamide (Cy)-induced tumor rejection. In growing tumors, tumor infiltrating macrophages (TIMs) produced interleukin (IL)-10. Beginning 6 h after Cy-treatment T cells in the tumor were inactivated and TIMs switched to interferon (IFN)-γ production. Both, IL-10 production before and IFN-γ production after Cy-treatment by TIMs required T cells. With the same kinetics as TIMs started to produce IFN-γ the tumor vasculature was destroyed which required IFN-γ receptor expression on host but not tumor cells. These events preceded hemorrhagic necrosis and residual tumor cell elimination by T cells. Together, T cells regulate the function of TIMs and tumor rejection can be induced by disturbing the stroma network.
CD4+ T Cells Regulate Surgical and Postinfectious Adhesion Formation
Authors: Chung Doo Ryeon , Chitnis Tanuja , Panzo Ronald J. , Kasper Dennis L. , Sayegh Mohamed H. , Tzianabos Arthur O.
Published: July 2002

The development of adhesions in the peritoneal and pelvic cavities, which commonly form after surgery or infection, cause significant morbidity and mortality. However, the pathogenesis of adhesion formation is still poorly understood. Because T cells are important in orchestrating fibrinogenic tissue disorders, we hypothesized that they play a critical role in the pathogenesis of peritoneal adhesion formation. Using a cecal abrasion surgical model in rodents, T cell depletion and adoptive transfer experiments demonstrated that this host response is dependent on CD4+ αβ T cells. These cells were also critical to adhesion formation associated with experimental intraabdominal sepsis. T cell transfer studies with mice deficient in signal transducer and activator of transcription (Stat)4 and Stat6 revealed that adhesion formation was dependent on a T helper 1 response. Activated T cells homed to the peritoneal cavity 6 hours after cecal abrasion surgery and predominated at this site during adhesiogenesis. Increased levels of the T cell–derived proinflammatory cytokine interleukin (IL)-17 and of neutrophil chemoattractant CXC chemokines macrophage inflammatory protein-2/CXCL8 and cytokine-induced neutrophil chemoattractant/CXCL1 were associated with adhesion formation. The production of these chemokines was dependent on T cells. Furthermore, the administration of neutralizing antibodies specific for IL-17 or the receptor that binds these CXC chemokines, CXC chemokine receptor 2, significantly reduced the degree of adhesion formation. These results demonstrate for the first time that the immunopathogenesis of adhesion formation is under the control of T cells and that T cell–derived cytokines and chemokines play important roles in the development of this deleterious host response.
Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors
Authors: Suda Norio , Franzius Dorothee , Fleig Andrea , Nishimura Seiichiro , Bödding Matthias , Hoth Markus , Takeshima Hiroshi , Penner Reinhold
Published: July 2002

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca2+ channels, we expressed α2, β, and γ DHPR subunits and a chimeric DHPR α1 subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca2+ transient (CaT) in the absence of extracellular Ca2+ ([Ca2+]o). However, in the presence of [Ca2+]o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca2+ concentration ([Ca2+]i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+]i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ channels, whereas the second component resulted from Ca2+ release through the RyR expressed in the membrane of internal Ca2+ stores. However, the onset and the rate of Ca2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca2+ current. These results suggest that the skeletal muscle RyR isoform supports Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.