bluuebunny/crossref_metadata_2025 huggingface dataset:
Retrograde trans-synaptic transfer of green fluorescent protein allows the genetic mapping of neuronal circuits in transgenic mice
Authors: Maskos Uwe , Kissa Karima , St. Cloment Cécile , Brûlet Philippe
Published: July 2002

The function of the nervous system is a consequence of the intricate synaptic connectivity of its neurons. Our understanding of these highly complex networks has profited enormously from methods used over the past two decades that are based on the mechanical injection of tracer molecules into brain regions. We have developed a genetic system for the mapping of synaptic connections during development of the mammalian central nervous system and in the mature brain. It is based on the transsynaptic transfer of green fluorescent protein (GFP) in the brains of mice using a fusion protein with a nontoxic fragment of tetanus toxin (TTC) expressed in defined neurons. These transgenic mice allowed us to visualize neurons, at single-cell resolution, that are in synaptic contact by the detection of GFP in interconnected circuits. Targeted genetic expression with a specific promoter permitted us to transfer GFP to defined subsets of neurons and brain regions. GFP–TTC is coexpressed with a
lacZ
reporter gene to discriminate neurons that produce the tracer from cells that have acquired it transneuronally. The marker shows selective transfer in the retrograde direction. We have used electron microscopic detection of GFP to define the ultrastructural features of the system. Our work opens up a range of possibilities for brain slice and
in vivo
studies taking advantage of the fluorescence of GFP. We point the way toward the use of powerful multiphoton technology and set the stage for the transsynaptic transfer of other proteins in the brains of mice.
The metal reductase activity of some multiheme cytochromes
c
: NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome
c
7
Authors: Assfalg Michael , Bertini Ivano , Bruschi Mireille , Michel Caroline , Turano Paola
Published: July 2002

The redox reaction between Cr
O

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and the fully reduced three-heme cytochrome
c
7
from
Desulfuromonas acetoxidans
to give chromium(III) and the fully oxidized protein has been followed by NMR spectroscopy. The hyperfine coupling between the oxidized protein protons and chromium(III), which remains bound to the protein, gives rise to line-broadening effects on the NMR resonances that can be transformed into proton-metal distance restraints. Structure calculations based on these unconventional constraints allowed us to demonstrate that chromium(III) binds at a unique site and to locate it on the protein surface. The metal ion is located 7.9 ± 0.4 Å from the iron of heme IV, 16.3 ± 0.7 Å from the iron of heme III, and 22.5 ± 0.5 Å from the iron of heme I. Shift changes caused by the presence of unreactive Mo
O

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, a Cr
O

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analogue, indicate the involvement of the same protein area in the anion binding. The titration of the oxidation of cytochrome
c
7
shows a detailed mechanism of action. The presence of a specific binding site supports the hypothesis of the biological role of this cytochrome as a metal reductase.
Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death
Authors: Jablonka Sibylle , Holtmann Bettina , Meister Gunter , Bandilla Michael , Rossoll Wilfried , Fischer Utz , Sendtner Michael
Published: July 2002

Neuronal degeneration in spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. SMN and the tightly interacting Gemin2 form part of a macromolecular complex (SMN complex) that mediates assembly of spliceosomal small nuclear ribonucleoproteins (U snRNPs). We used mouse genetics to investigate the function of this complex in motoneuron maintenance. Reduced Smn/Gemin2 protein levels lead to disturbed U snRNP assembly as indicated by reduced nuclear accumulation of Sm proteins. This finding correlates with enhanced motoneuron degeneration in
Gemin2
+/−
/
Smn
+/−
mice. Our data provide
in vivo
evidence that impaired production of U snRNPs contributes to motoneuron degeneration.
Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover
Authors: Street John , Bao Min , deGuzman Leo , Bunting Stuart , Peale Franklin V. , Ferrara Napoleone , Steinmetz Hope , Hoeffel John , Cleland Jeffrey L. , Daugherty Ann , van Bruggen Nicholas , Redmond H. Paul , Carano Richard A. D. , Filvaroff Ellen H.
Published: July 2002

Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.
Short RNA duplexes produced by hydrolysis with
Escherichia coli
RNase III mediate effective RNA interference in mammalian cells
Authors: Yang Dun , Buchholz Frank , Huang Zhongdong , Goga Andrei , Chen Chih-Ying , Brodsky Frances M. , Bishop J. Michael
Published: July 2002

Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of
Escherichia coli
RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in
Drosophila
S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.
Molecular portraits of B cell lineage commitment
Authors: Müschen Markus , Lee Sanggyu , Zhou Guolin , Feldhahn Niklas , Barath Varun Singh , Chen Jianjun , Moers Cordula , Krönke Martin , Rowley Janet D. , Wang San Ming
Published: July 2002

In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34
+
HSCs and CD10
+
CD19
+
PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might reflect selection for the expression of a functional pre-BCR within the bone marrow. Besides known regulator genes of early B cell development such as
PAX5
,
E2A
, and
EBF
, the most abundantly expressed genes in PBCs include
ATM
,
PDGFRA
,
SIAH1
,
PIM2
,
C/EBPB
,
WNT16
, and
TCL1
, the role of which has not been established yet in early B cell development.
A crucial role for the putative
Arabidopsis
topoisomerase VI in plant growth and development
Authors: Yin Yanhai , Cheong Hyeonsook , Friedrichsen Danielle , Zhao Yunde , Hu Jianping , Mora-Garcia Santiago , Chory Joanne
Published: July 2002

Plant steroid hormones, brassinosteroids (BRs), play important roles throughout plant growth and development. Plants defective in BR biosynthesis or perception display cell elongation defects and severe dwarfism. Two dwarf mutants named
bin3
and
bin5
with identical phenotypes to each other display some characteristics of BR mutants and are partially insensitive to exogenously applied BRs. In the dark,
bin3
or
bin5
seedlings are de-etiolated with short hypocotyls and open cotyledons. Light-grown mutant plants are dwarfs with short petioles, epinastic leaves, short inflorescence stems, and reduced apical dominance. We cloned
BIN3
and
BIN5
and show that BIN5 is one of three putative
Arabidopsis
SPO11 homologs (AtSPO11-3) that also shares significant homology to archaebacterial topoisomerase VI (TOP6) subunit A, whereas BIN3 represents a putative eukaryotic homolog of TOP6B. The pleiotropic dwarf phenotypes of
bin5
establish that, unlike all of the other SPO11 homologs that are involved in meiosis, BIN5/AtSPO11-3 plays a major role during somatic development. Furthermore, microarray analysis of the expression of about 5500 genes in
bin3
or
bin5
mutants indicates that about 321 genes are down-regulated in both of the mutants, including 18 of 30 BR-induced genes. These results suggest that BIN3 and BIN5 may constitute an
Arabidopsis
topoisomerase VI that modulates expression of many genes, including those regulated by BRs.
The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in
Arabidopsis
Authors: He Jun-Xian , Gendron Joshua M. , Yang Yanli , Li Jianming , Wang Zhi-Yong
Published: July 2002

Brassinosteroids (BRs) are a class of steroid hormones essential for normal growth and development in plants. BR signaling involves the cell-surface receptor BRI1, the glycogen synthase kinase-3-like kinase BIN2 as a negative regulator, and nuclear proteins BZR1 and BZR2/BES1 as positive regulators. The interactions among these components remain unclear. Here we report that BRs induce dephosphorylation and accumulation of BZR1 protein. Experiments using a proteasome inhibitor, MG132, suggest that phosphorylation of BZR1 increases its degradation by the proteasome machinery. BIN2 directly interacts with BZR1 in yeast two-hybrid assays, phosphorylates BZR1
in
vitro
, and negatively regulates BZR1 protein accumulation
in
vivo
. These results strongly suggest that BIN2 phosphorylates BZR1 and targets it for degradation and that BR signaling causes BZR1 dephosphorylation and accumulation by inhibiting BIN2 activity.
The emergence of humans: The coevolution of intelligence and longevity with intergenerational transfers
Authors: Kaplan Hillard S. , Robson Arthur J.
Published: July 2002

Two striking differences between humans and our closest living relatives, chimpanzees and gorillas, are the size of our brains (larger by a factor of three or four) and our life span (longer by a factor of about two). Our thesis is that these two distinctive features of humans are products of coevolutionary selection. The large human brain is an investment with initial costs and later rewards, which coevolved with increased energy allocations to survival. Not only does this theory help explain life history variation among primates and its extreme evolution in humans; it also provides new insight into the evolution of longevity in other biological systems. We introduce and apply a general formal demographic model for constrained growth and evolutionary tradeoffs in the presence of life-cycle transfers between age groups in a population.
Extent of digestion affects the success of amplifying human DNA from blood meals of Anopheles gambiae (Diptera: Culicidae)
Authors: Mukabana W.R. , Takken W. , Seda P. , Killeen G.F. , Hawley W.A. , Knols B.G.J.
Published: July 2002

The success of distinguishing blood meal sources of Anopheles gambiae Giles through deoxyribonucleic acid (DNA) profiling was investigated by polymerase chain reaction (PCR) amplification at the TC-11 and VWA human short tandem repeats (STR) loci. Blood meal size and locus had no significant effect on the success of amplifying human DNA from blood meals digested for 0, 8, 16, 24 and 32 h (P = 0.85 and 0.26 respectively). However, logistic regression found a significant negative relationship between time since ingestion and the success probability of obtaining positive PCR products among meals digested for between 8 and 32 h (P = 0.001). Approximately 80% of fresh blood meals were successfully profiled. After 8 h, the proportion of blood meals that could be successfully profiled decreased slowly with time after ingestion, dropping to below 50% after approximately 15 h. There was no significant difference in the success of amplifying human DNA from blood meals of mosquitoes killed at time 0 and 8 h after ingestion (P = 0.272).
Overseeing organizations: configuring action and its environment
Authors: Heath Christian , Luff Paul , Svensson Marcus Sanchez
Published: July 2002

Despite the widespread deployment of CCTV through most major cities and towns in great Britain, and the importance of surveillance to contemporary debates within the social sciences, there remains relatively little detailed research concerned with the practical use of these technologies in the workplace. In this paper, we examine how personnel in the operation rooms in London Underground use CCTV and related equipment to identify problems and events and to develop a co‐ordinated response. In particular, we consider how personnel configure scenes to make sense of and interpret the conduct of the travelling public in organizationally relevant ways, and how they shape the ways in which both passengers and staff see and respond to each others’ actions. In addressing how personnel constitute the sense and significance of CCTV images, we reflect on the development of information processing systems which are designed to automatically detect conduct and events.
Gaspirali v. Il'Minskii: Two Identity Projects for the Muslims of the Russian Empire
Authors: Özgür Tuna Mustafa
Published: July 2002

In 1913, an article in a Russian missionary journal compared two “very typical representatives” of Islamic studies in Russia: İsmail Bey Gaspıralı (1851–1914) and Nikolai Ivanovich Il'minskii (1822–1891). Nothing could better symbolize the two opposing points of view about the past, present and future of the Muslims of Russia in 1913. Il'minskii was a Russian Orthodox missionary whose ideas and efforts had formed the imperial perceptions and policies about the Muslims of the Russian empire in the late Tsarist period, while Gaspıralı was a Muslim educator and publisher whose ideas and efforts had shaped the Muslim society per se in the same period. Il'minskii, beginning in the 1860s, and Gaspıralı, beginning in the 1880s, developed two formally similar but inherently contradictory programs for the Muslims of the Russian empire. Schooling and the creation of a literary language or literary languages constituted the hearts of both of their programs. Besides their own efforts, both Gaspıralı and Il'minskii had a large number of followers that diligently worked to put their programs into practice among the Muslims of Russia. As a result of the inherent contradiction of these programs, a bitter controversy developed between what we may call the Il'minskii and Gaspıralı groups, which particularly intensified after the revolution of 1905. In this article, I will discuss the underlying causes and development of this controversy by focusing on the role of language in the programs of Gaspıralı and Il'minskii. Then, I will conclude my article with an evaluation of the legacies of these two individuals in their own time and beyond.
Covert orienting to the locations of targets and distractors: Effects on response channel activation in a flanker task
Authors: Ro Tony , Machado Liana , Kanwisher Nancy , Rafal Robert D.
Published: July 2002

The role of covert orienting of attention in response channel activation was examined using the flanker interference and precueing paradigms. Four experiments assessed the influence of distractors on the discrimination of a target colour patch under cueing conditions (three with non-informative, exogenous cues and one with informative, endogenous cues) that modulated attention at the flanker or target locations. Across all of the experiments, the amount of interference generated by the distractors was not modulated by the facilitation and inhibition of return induced by spatial attention precues. These results are consistent with previous reports of patients with neglect, which demonstrated that flanker interference proceeds at unattended locations (Audet, Bub, & Lecours, 1991; Cohen, Ivry, Rafal, & Kohn, 1995), and they suggest that response channel activation can occur independently from spatial attention.
Head-centred meridian effect on auditory spatial attention orienting
Authors: Ferlazzo Fabio , Couyoumdjian Alessandro , Padovani Tullia , Belardinelli Marta Olivetti
Published: July 2002

Six experiments examined the issue of whether one single system or separate systems underlie visual and auditory orienting of spatial attention. When auditory targets were used, reaction times were slower on trials in which cued and target locations were at opposite sides of the vertical head-centred meridian than on trials in which cued and target locations were at opposite sides of the vertical visual meridian or were not separated by any meridian. The head-centred meridian effect for auditory stimuli was apparent when targets were cued by either visual (Experiments 2, 3, and 6) or auditory cues (Experiment 5). Also, the head-centred meridian effect was found when targets were delivered either through headphones (Experiments 2, 3, and 5) or external loudspeakers (Experiment 6). Conversely, participants showed a visual meridian effect when they were required to respond to visual targets (Experiment 4). These results strongly suggest that auditory and visual spatial attention systems are indeed separate, as far as endogenous orienting is concerned.
Searching for threat
Authors: Tipples Jason , Young Andrew W. , Quinlan Philip , Broks Paul , Ellis Andrew W.
Published: July 2002

In a series of experiments, a visual search task was used to test the idea that biologically relevant threatening stimuli might be recognized very quickly or capture visuo-spatial attention. In Experiment 1, there was evidence for both faster detection and faster search rates for threatening animals than for plants. However, examination of the basis of this effect in Experiment 2 showed that it was not due to threat per se, as detection and search rate advantages were found for pleasant rather than threatening animals compared to plants. In Experiment 3, participants searched for the plants and pleasant and threatening animals used in Experiments 1 and 2, among a fixed heterogeneous selection of non-target items. There was no search rate or detection advantage for threatening animals compared to pleasant animals or plants. The same targets and non-targets as those used in Experiment 3 were also used in Experiment 4. In Experiment 4, participants searched for targets that were presented either close to or distant from an initial fixation point. There was no evidence for a “threat” detection advantage either close to or distant from the cross. Finally, an experiment was conducted in which target categories (fruit, flowers, and animals) were not pre-specified prior to each trial block. There were no differences in reaction times to detect pleasant animals, threatening animals, or fruit. We conclude that the visual search paradigm does not readily reveal any biases that might exist for threatening stimuli in the general population.
Extinction of conditioned taste aversions: Effects of concentration and overshadowing
Authors: Taylor Kathryn M. , Boakes Robert A.
Published: July 2002

Extinction of conditioned taste aversions was examined as a function of taste concentration and of the presence of an additional taste. The results of Experiment 1 were consistent with previous evidence in that a conditioned aversion to high concentration saline was more persistent in extinction than an aversion to a low concentration. However, when floor effects were avoided the rate of extinction was faster for the higher (1%) concentration than for 0.2% saline (Experiment 2), a result consistent with accounts of extinction in other preparations. Three further experiments examined extinction of a conditioned sucrose aversion. The addition of 1% saline, but not of 0.2% saline, to sucrose during extinction produced overshadowing (“protection from extinction”; Experiment 3). Such overshadowing by saline was detected after two, but not after a single extinction trial (Experiment 4). This last finding suggests that under the conditions of the present experiments sweet and salty tastes function as elemental stimuli competing for loss of associative strength. No overshadowing was found when almond (an aqueous odour) was used in place of saline as the added stimulus, even when high concentrations of almond were used that produced observable neophobia (Experiments 5A and 5B).
John Bates Clark's Defense of Mandatory Arbitration and Minimum Wage Legislation
Authors: Prasch Robert E.
Published: July 2002

The ideal of Political Economy is not unrestricted competition, but competition that is truly free, because controlled by justice and by law. The distinction between freedom and license needs to be preserved in this department of political philosophy. With that distinction clearly maintained, we may still retain, in economics as in politics, our beautiful watchword, liberty (John Bates Clark 1879, p. 167).

John Bates Clark has had a formidable impact on the development of economic theory and the theory of income distribution. In addition, Clark's marginal product theory of distribution has often played an instrumental role in the defense oflaissez-fairepolicies. His theory has been used to criticize a wide variety of market interventions, including minimum wage legislation. As a result it is not surprising that Clark's theory of income distribution, and Clark himself, have drawn more than a few critics.
Keynes and Harrod on the Classical Theory of Interest: More on the Origin of the Only Diagram in the General Theory
Authors: Besomi Daniele
Published: July 2002

Rod O'Donnell's (1999a, 1999b) and James Ahiakpor's (1999) contributions on the origin of the only diagram in J. M. Keynes's General Theory raise some interesting points regarding the use of documentary evidence for interpreting authors' ideas or specific passages in their writings.
Europe and European culture - a conceptual analysis1
Authors: Ifversen Jan
Published: July 2002

Current debates on Europe and European culture illustrate the growing difficulties in agreeing on the meaning of Europe. More than ever, Europe is a contested concept. Through a conceptual analysis, this article outlines different positions taken in filling the concept of Europe with meaning. The focus is on Europe as a culture. In line with modern cultural theory, articulations of Europe as a culture may be viewed as displaced within a gamut going from the most essentialist to the most constructivist. Essentialist articulations of European culture typically follow the traditional model of culture as it has been fixed through the nation-states. As is shown in the article, attempts of articulating a European culture along the lines of nations run into conceptual troubles. Recently, a more constructivist mood has come to dominate the ways Europe can be given meaning. A shift from culture to identity has marked this new constructivism. But constructivism also points in the direction of a political field where culture is challenged by a political vocabulary. This article demonstrates the different transfers from a cultural to a political field in current statements on European culture.
Changes in status attainment in Hungary between 1910 and 1989
Authors: Luijkx Ruud , Róbert Péter , Graaf Paul M. De , Ganzeboom Harry B.G.
Published: July 2002

This article addresses the effects of economic and political change on social mobility in Hungary between 1910 and 1989 by investigating whether the effects of family background on schooling and the effects of family background and schooling on first occupation vary between periods in Hungary's twentieth-century history. For this purpose, we distinguish five periods: the long-lasting Depression (1910 to 1933), the period around the Second World War (1934 to 1948), the long 1950s (1949 to 1967), the period of reform socialism (1968 to 1982), and the decline of socialism (1983 to 1989). Using large-scale datasets from 1973, 1983, 1992 and 1993, we are able to investigate developments in the parameters of the status attainment model for about 75,000 men and women. We use spline regressions to find out whether trends in the effects vary between periods. Linear secular trends in the effects of family background and schooling do not predominate; spline models reveal discontinuities between periods. On the other hand, a trend from ascription to achievement both for men and women can be observed. In contrast to the general assumption, the most important deviation from the general trend has taken place in the years before the communist take-over.
Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor
Authors: Hunyady László , Baukal Albert J. , Gáborik Zsuzsanna , Olivares-Reyes Jesus A. , Bor Márta , Szaszák Márta , Lodge Robert , Catt Kevin J. , Balla Tamas
Published: July 2002

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.
Dok-related protein negatively regulates T cell development via its RasGTPase-activating protein and Nck docking sites
Authors: Gugasyan Raffi , Quilici Cathy , I Stacey T.T. , Grail Dianne , Verhagen Anne M. , Roberts Andrew , Kitamura Toshio , Dunn Ashley R. , Lock Peter
Published: July 2002

Downstream of kinase (Dok)–related protein (DokR, also known as p56dok/FRIP/Dok-R) is implicated in cytokine and immunoreceptor signaling in myeloid and T cells. Tyrosine phosphorylation induces DokR to bind the signal relay molecules, RasGTPase-activating protein (RasGAP) and Nck. Here, we have examined the function of DokR during hematopoietic development and the requirement for RasGAP and Nck binding sites in its biological function. Retroviral-mediated expression of DokR in bone marrow cells dramatically inhibited their capacity to form colonies in vitro in response to the cytokines macrophage colony–stimulating factor and stem cell factor, whereas responses to interleukin-3 and granulocyte macrophage colony–stimulating factor were only weakly affected. When introduced into lethally irradiated mice, hematopoietic cells expressing DokR showed a drastically reduced capacity to repopulate lymphoid tissues. Most notably, DokR dramatically reduced repopulation of the thymus, in part by reducing the number of T cell precursors seeding in the thymus, but equally, through inhibiting the transition of CD4−CD8− to CD4+CD8+ T cells. Consequently, the number of mature peripheral T cells was markedly reduced. In contrast, a minimal effect on B cell and myeloid lineage development was observed. Importantly, functional RasGAP and Nck binding sites were found to be essential for the biological effects of DokR in vitro and in vivo.
Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes
Authors: Barreiro Olga , Yáñez-Mó María , Serrador Juan M. , Montoya María C. , Vicente-Manzanares Miguel , Tejedor Reyes , Furthmayr Heinz , Sánchez-Madrid Francisco
Published: July 2002

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.
Effects of cell tension on the small GTPase Rac
Authors: Katsumi Akira , Milanini Julie , Kiosses William B. , del Pozo Miguel A. , Kaunas Roland , Chien Shu , Hahn Klaus M. , Schwartz Martin Alexander
Published: July 2002

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.
Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells
Authors: Salmon Wendy C. , Adams Michael C. , Waterman-Storer Clare M.
Published: July 2002

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.
Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation
Authors: Li Shaohua , Harrison David , Carbonetto Salvatore , Fässler Reinhard , Smyth Neil , Edgar David , Yurchenco Peter D.
Published: July 2002

Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for γ1-laminin, β1-integrin and α/β-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables β1-integrin–null embryoid bodies to assemble basement membrane and achieve epiblast with β1-integrin enabling expression of the laminin α1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via β1-integrin through LG1–3 and requiring polymerization, can regulate dystroglycan expression.
Transcriptome profiling to identify genes involved in peroxisome assembly and function
Authors: Smith Jennifer J. , Marelli Marcello , Christmas Rowan H. , Vizeacoumar Franco J. , Dilworth David J. , Ideker Trey , Galitski Timothy , Dimitrov Krassen , Rachubinski Richard A. , Aitchison John D.
Published: July 2002

Yeast cells were induced to proliferate peroxisomes, and microarray transcriptional profiling was used to identify PEX genes encoding peroxins involved in peroxisome assembly and genes involved in peroxisome function. Clustering algorithms identified 224 genes with expression profiles similar to those of genes encoding peroxisomal proteins and genes involved in peroxisome biogenesis. Several previously uncharacterized genes were identified, two of which, YPL112c and YOR084w, encode proteins of the peroxisomal membrane and matrix, respectively. Ypl112p, renamed Pex25p, is a novel peroxin required for the regulation of peroxisome size and maintenance. These studies demonstrate the utility of comparative gene profiling as an alternative to functional assays to identify genes with roles in peroxisome biogenesis.
On the Role of Dendritic Cells in Peripheral T Cell Tolerance and Modulation of Autoimmunity
Authors: Legge Kevin L. , Gregg Randal K. , Maldonado-Lopez Roberto , Li Lequn , Caprio Jacque C. , Moser Muriel , Zaghouani Habib
Published: July 2002

Recently, it has become clear that dendritic cells (DCs) are essential for the priming of T cell responses. However, their role in the maintenance of peripheral T cell tolerance remains largely undefined. Herein, an antigen-presenting cell (APC) transfer system was devised and applied to experimental allergic encephalomyelitis (EAE), to evaluate the contribution that DCs play in peripheral T cell tolerance. The CD8α−CD4+ subset, a minor population among splenic DCs, was found to mediate both tolerance and bystander suppression against diverse T cell specificities. Aggregated (agg) Ig-myelin oligodendrocyte glycoprotein (MOG), an Ig chimera carrying the MOG 35–55 peptide, binds and cross-links FcγR on APC leading to efficient peptide presentation and interleukin (IL)-10 production. Furthermore, administration of agg Ig-MOG into diseased mice induces relief from clinical EAE involving multiple epitopes. Such recovery could not occur in FcγR-deficient mice where both uptake of Ig-MOG and IL-10 production are compromised. However, reconstitution of these mice with DC populations incorporating the CD8α−CD4+ subset restored Ig-MOG–mediated reversal of EAE. Transfer of CD8α+ or even CD8α−CD4− DCs had no effect on the disease. These findings strongly implicate DCs in peripheral tolerance and emphasize their functional potency, as a small population of DCs was able to support effective suppression of autoimmunity.
Activation and Differentiation of Autoreactive B-1 Cells by Interleukin 10 Induce Autoimmune Hemolytic Anemia in Fas-deficient Antierythrocyte Immunoglobulin Transgenic Mice
Authors: Watanabe Norihiko , Ikuta Koichi , Nisitani Sazuku , Chiba Tsutomu , Honjo Tasuku
Published: July 2002

The Fas (CD95) gene is among critical genetic factors in some autoimmune diseases, which are characterized by autoantibody (autoAb) productions. In mice, mutations in the Fas gene cause lymphoproliferation (lpr) which predominantly develops glomerulonephritis, whereas the mutations in human cause autoimmune lymphoproliferative syndrome (ALPS) characterized by autoimmune hemolytic anemia (AIHA) and thrombocytopenia. Although the mechanism of antinuclear Ab in Fas-deficient background has been well characterized, that of antierythrocyte Ab production in ALPS has been still unclear. To investigate this mechanism, we developed a mouse line by crossing the antierythrocyte antibody transgenic mice (H+L6 mice) and Fas-deficient mice. Although Fas deficiency did not break tolerance of autoreactive B-2 cells in H+L6 mice, autoreactive B-1 cells in Fas-deficient H+L6 homozygous mice became activated and differentiated into autoAb-producing cells in mesenteric lymph nodes and lamina propria of intestine, resulting in severe anemia. In addition, serum levels of interleukin (IL)-10 significantly increased in Fas−/− × H+L6 homozygous mice and administration of anti–IL-10 Ab prevented exacerbation of autoAb production and AIHA. These results suggest that activation of B-1 cells is responsible for induction of AIHA in Fas-deficient condition and that IL-10 plays a critical role in terminal differentiation of B-1 cells in these mice.
Mesenchymal Stem Cells in Perichondrium Express Activated Leukocyte Cell Adhesion Molecule and Participate in Bone Marrow Formation
Authors: Arai Fumio , Ohneda Osamu , Miyamoto Takeshi , Zhang Xiu Qin , Suda Toshio
Published: July 2002

Perichondrium in fetal limb is composed of undifferentiated mesenchymal cells. However, the multipotency of cells in this region and the role of perichondrium in bone marrow formation are not well understood. In this report, we purified and characterized perichondrial cells using a monoclonal antibody against activated leukocyte cell adhesion molecule (ALCAM) and investigated the role of perichondrial cells in hematopoietic bone marrow formation. ALCAM is expressed on hematopoietic cells, endothelial cells, bone marrow stromal cells, and mesenchymal stem cells and mediates homophilic (ALCAM–ALCAM)/heterophilic (ALCAM-CD6) cell adhesion. Here we show by immunohistochemical staining that ALCAM is expressed in perichondrium. ALCAM+ perichondrial cells isolated by FACS® exhibit the characteristics of mesenchymal stem cells. ALCAM+ cells can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, which can support osteoclastogenesis, hematopoiesis, and angiogenesis. Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited. Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels. These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow.
Implications of Phase Variation of a Gene (pgtA) Encoding a Pilin Galactosyl Transferase in Gonococcal Pathogenesis
Authors: Banerjee Asesh , Wang Rong , Supernavage Sherry L. , Ghosh Salil K. , Parker James , Ganesh Nisha F. , Wang Peng G. , Gulati Sunita , Rice Peter A.
Published: July 2002

The pilin glycoprotein (PilE) is the main building block of the pilus of Neisseria gonorrhoeae (gonococcus [GC]). GC pilin is known to carry a disaccharide O-glycan, which has an αGal attached to the O-linked GlcNAc by a 1–3 glycosidic bond. In this report, we describe the cloning and characterization of the GC gene, pilus glycosyl transferase A (pgtA), which encodes the galactosyl transferase that catalyzes the synthesis of this Gal–GlcNAc bond of pilin glycan. A homopolymeric tract of Gs (poly-G) is present in the pgtA gene of many GC strains, and this pgtA with poly-G can undergo phase variation (Pv). However, in many other GC, pgtA lacks the poly-G and is expressed constitutively without Pv. Furthermore, by screening a large number of clinical isolates, a significant correlation was observed between the presence of poly-G in pgtA and the dissemination of GC infection. Poly-G was found in pgtA in all (24 out of 24) of the isolates from patients with disseminated gonococcal infection (DGI). In contrast, for the vast majority (20 out of 28) of GC isolated from uncomplicated gonorrhea (UG) patients, pgtA lacked the poly-G. These results indicate that Pv of pgtA is likely to be involved in the conversion of UG to DGI.
Multidrug Efflux Systems Play an Important Role in the Invasiveness of Pseudomonas aeruginosa
Authors: Hirakata Yoichi , Srikumar Ramakrishnan , Poole Keith , Gotoh Naomasa , Suematsu Takashi , Kohno Shigeru , Kamihira Shimeru , Hancock Robert E. W. , Speert David P.
Published: July 2002

Pseudomonas aeruginosa is an important opportunistic human pathogen. Certain strains can transmigrate across epithelial cells, and their invasive phenotype is correlated with capacity to cause invasive human disease and fatal septicemia in mice. Four multidrug efflux systems have been described in P. aeruginosa, however, their contribution to virulence is unclear. To clarify the role of efflux systems in invasiveness, P. aeruginosa PAO1 wild-type (WT) and its efflux mutants were evaluated in a Madin-Darby canine kidney (MDCK) epithelial cell monolayer system and in a murine model of endogenous septicemia. All efflux mutants except a ΔmexCD-oprJ deletion demonstrated significantly reduced invasiveness compared with WT. In particular, a ΔmexAB-oprM deletion strain was compromised in its capacity to invade or transmigrate across MDCK cells, and could not kill mice, in contrast to WT which was highly invasive (P < 0.0006) and caused fatal infection (P < 0.0001). The other mutants, including ΔmexB and ΔmexXY mutants, were intermediate between WT and the ΔmexAB-oprM mutant in invasiveness and murine virulence. Invasiveness was restored to the ΔmexAB-oprM mutant by complementation with mexAB-oprM or by addition of culture supernatant from MDCK cells infected with WT. We conclude that the P. aeruginosa MexAB-OprM efflux system exports virulence determinants that contribute to bacterial virulence.
Induction of NFATc2 Expression by Interleukin 6 Promotes T Helper Type 2 Differentiation
Authors: Diehl Sean , Chow Chi-Wing , Weiss Linda , Palmetshofer Alois , Twardzik Thomas , Rounds Laura , Serfling Edgar , Davis Roger J. , Anguita Juan , Rincón Mercedes
Published: July 2002

Interleukin (IL)-6 is produced by professional antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been previously shown that APC-derived IL-6 promotes the differentiation of naive CD4+ T cells into effector T helper type 2 (Th2) cells. Here, we have studied the molecular mechanism for IL-6–mediated Th2 differentiation. During the activation of CD4+ T cells, IL-6 induces the production of IL-4, which promotes the differentiation of these cells into effector Th2 cells. Regulation of IL-4 gene expression by IL-6 is mediated by nuclear factor of activated T cells (NFAT), as inhibition of NFAT prevents IL-6–driven IL-4 production and Th2 differentiation. IL-6 upregulates NFAT transcriptional activity by increasing the levels of NFATc2. The ability of IL-6 to promote Th2 differentiation is impaired in CD4+ T cells that lack NFATc2, demonstrating that NFATc2 is required for regulation of IL-4 gene expression by IL-6. Regulation of NFATc2 expression and NFAT transcriptional activity represents a novel pathway by which IL-6 can modulate gene expression.