bluuebunny/crossref_metadata_2025 huggingface dataset:
d
-Serine is an endogenous ligand for the glycine site of the
N
-methyl-
d
-aspartate receptor
Authors: Mothet Jean-Pierre , Parent Angèle T. , Wolosker Herman , Brady Roscoe O. , Linden David J. , Ferris Christopher D. , Rogawski Michael A. , Snyder Solomon H.
Published: July 2002

Functional activity of
N
-methyl-
d
-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although
d
-serine is a more potent agonist. Localizations of
d
-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of
d
-serine with
d
-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch–clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied
d
-serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus,
d
-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.
A default mode of brain function
Authors: Raichle Marcus E. , MacLeod Ann Mary , Snyder Abraham Z. , Powers William J. , Gusnard Debra A. , Shulman Gordon L.
Published: July 2002

A baseline or control state is fundamental to the understanding of
most complex systems. Defining a baseline state in the human brain,
arguably our most complex system, poses a particular challenge. Many
suspect that left unconstrained, its activity will vary unpredictably.
Despite this prediction we identify a baseline state of the normal
adult human brain in terms of the brain oxygen extraction fraction or
OEF. The OEF is defined as the ratio of oxygen used by the brain to
oxygen delivered by flowing blood and is remarkably uniform in the
awake but resting state (e.g., lying quietly with eyes closed). Local
deviations in the OEF represent the physiological basis of signals of
changes in neuronal activity obtained with functional MRI during a wide
variety of human behaviors. We used quantitative metabolic and
circulatory measurements from positron-emission tomography to obtain
the OEF regionally throughout the brain. Areas of activation were
conspicuous by their absence. All significant deviations from the mean
hemisphere OEF were increases, signifying deactivations, and resided
almost exclusively in the visual system. Defining the baseline state of
an area in this manner attaches meaning to a group of areas that
consistently exhibit decreases from this baseline, during a wide
variety of goal-directed behaviors monitored with positron-emission
tomography and functional MRI. These decreases suggest the existence of
an organized, baseline default mode of brain function that is suspended
during specific goal-directed behaviors.
Residential Profiles of Surinamese and Moroccans in Amsterdam
Authors: Deurloo Rinus , Musterd Sako
Published: July 2002

At present, politicians in the Netherlands tend to pay more attention to bridging cultural gaps between various ethnic groups than reducing class differences. In that context, it is frequently assumed that ethnic integration processes can be facilitated by establishing a mixed population. In order to reach that objective, municipalities and local housing associations are encouraged to realise heterogeneous residential areas. However, this kind of policy rests on the assumption of a clear relationship between a spatial mix of the population in ethnic terms and the level of integration in society. The validity of this assumption is challenged in this paper in its investigation of housing profiles for areas containing varying immigrant densities in Amsterdam. Micro-level spatial data for all (over 16 000) postal-code areas in the city were compiled to substantiate this claim. The rich data-set allowed a detailed comparison to be made of the housing situation outside, within and between various ethnic group concentration areas which were constructed from the postal-code areas. This paper discusses the findings for Surinamese and Moroccans, the two largest non-native groups in Amsterdam. It is shown that, in general, all the residential neighbourhoods identified have heterogeneous housing profiles. The results suggest that other factors should be taken into account in order to understand the spatial segregation of immigrant groups. Such factors particularly include economic restructuring, the welfare state—especially redistributive mechanisms—urban history, general housing policy and cultural orientation.
Independent Technology-based Firms: The Perceived Benefits of a Science Park Location
Authors: Westhead Paul , Batstone Stephen
Published: July 2002

This study explores the perceived benefits of a science park location in the UK for independent technology-based firms. Property needs of independent science parks firms were compared with the property needs of a 'control' group of similar firms located off-park. Factors which influenced owner-managers to locate their ventures on a science park or an off-park location were ascertained. Further, the perceived benefits of selected locations were explored. Conclusions and implications for policy-makers and practitioners are detailed.
Ethnic Segregation in Cologne, Germany, 1984-94
Authors: Friedrichs Jürgen
Published: July 2002

The city of Cologne is, like Frankfurt/Main, Munich and Stuttgart, one of the German cities with high shares of ethnic minorities. In this paper, ethnic segregation in Cologne is analysed for three points in time: 1984, 1989 and 1994. One of the main conclusions is that segregation is declining for many groups, indicating a process of spatial dispersion across the city. 'New' immigrants, however, tend to be more segregated than 'older' groups. Economic conditions seem to be one of the crucial explaining elements. They influence the extent and development of segregation patterns directly, as well as indirectly.
Ethnic Clusters in Amsterdam, 1994-96: A Micro-area Analysis
Authors: Deurloo Marinus C. , Musterd Sako
Published: July 2002

Ghetto formation is a recurrent item in debates on urban futures all over the world, the Netherlands not excluded. Many people express their fear for extreme separation between ethnic groups. Many Europeans tend to believe that the recent growth in the number of immigrants in the larger cities will lead to ghetto-like developments, as has happened in US cities with regard to the black population. However, little evidence is available to support that idea. In this paper, this question is addressed based on micro-level spatial data for immigrant groups in Amsterdam. Data were available for two recent years, which provided the opportunity to study the dynamics of ethnic concentrations. Conclusions are, first, that the spatial segregation of immigrant groups in Amsterdam is far from the ghetto-type segregation in US cities; and secondly, that there are indications of the instability of ethnic concentrations, which is considered an indication against the development of ghettos as well. Characteristics of the Dutch welfare state may provide part of the explanation.
The Entrenchment of Urban Dispersion: Residential Preferences and Location Patterns in the Dispersed City
Authors: Filion Pierre , Bunting Trudi , Warriner Keith
Published: July 2002

The paper portrays three aspects of urban dispersion: urban structure, residents' location and land-use preferences, and social ecology. To explain the dynamic inherent in this form of urbanisation, it suggests an explanatory model concentrating on shifts in the respective importance of space, place and proximity associated with the passage from traditional monocentric to dispersed urban form. The paper draws its empirical substance from the Kitchener Census Metropolitan Area, one of the most dispersed metropolitan regions in Canada. The Kitchener case study highlights the defining characteristics of a dispersed urban structure: high automobile dependence, a scattering of origins and destinations, and a resulting absence of pronounced accessibility gradients. The paper also reports the results of a survey which indicates a harmonisation of residents' preferences with the main features of dispersion. The case study ends by mapping the residential location patterns of two groups with a disproportionate influence on new urban development: high income households and families with children. Their concentric distribution is consistent with survey results. In the light of the prevailing transport-land-use relation and of residents' location choices and expressed preferences, the paper foresees a further entrenchment of the dispersed urban structure. The paper closes by explaining the limited success of most intensification policies and by exploring the possibility of injecting more diversity into the dispersed landscape in order to accommodate a growing variety of lifestyles.
Local Commercial and Industrial Rental Trends and Property Market Constraints
Authors: Jones Colin , Orr Allison
Published: July 2002

The paper assesses the differential impact of property market constraints on the long-run rental trends in each of the commercial and industrial property market sectors. The perceived view that supply constraints are very inelastic in the retail sector, less so for offices and elastic for industrial properties is reconsidered. A series of hypotheses are derived and tested by an analysis of variance technique which decomposes rental change into local and national components. The paper concludes that changes in the pattern of spatial activity and specific property market processes have resculptured the nature of supply constraints. The result is that the spectrum of supply elasticities has narrowed.
Toxicology Studies with Recombinant Staphylokinase and with SY 161-P5, a Polyethylene Glycol-Derivatized Cysteine-Substitution Mutant
Authors: Moons Lieve , Vanlinthout Ingrid , Roelants Ivo , Moreadith Randall , Collen Desire , Rapold Hans J.
Published: July 2002

SY161-P5, a polyethylene glycol derivatized (PEGylated) mutant of the recombinant Staphylokinase (rSak) variant SakSTAR, exhibiting reduced antigenicity, is in clinical development for treatment of acute myocardial infarction as a single bolus injection. A series of safety studies were performed in vivo as a routine toxicology program with SY 161-P5 (PEG-rSakSTAR) and with the recombinant Staphylokinase variant Sak42D (rSak42D). For both compounds, intravenous single bolus injections of up to 100-fold therapeutic equivalent, as well as repeated injections during 7 to 28 days revealed no significant pathological findings in mice, rats or hamsters. However, New Zealand white rabbits developed clinically silent, multifocal myocarditis following single or repeat doses of SY 161-P5 or of Sak42D. These findings were dose-independent and reversible. A similar speciesspecific cardiotoxic effect has previously been described for other proteolytic proteins, including the approved drugs Streptokinase and Acetylated Plasminogen Streptokinase Complex (APSAC). The large experience with these drugs, as well as the clinical data accumulated both with PEGylated and non-PEGylated rSak variants to date, do not indicate cardiotoxic hazards associated with the use of these drugs in humans.
Morphological Classification of Dental Lesions Induced by Various Antitumor Drugs in Mice
Authors: Satoh Hiroshi , Uesugi Yasuo , Kawabata Takami , Mori Kazuhiko , Fuj Fumitaka , Kashimoto Yoshinori , Kajimura Tetsuyo , Furuhama Kazuhisa
Published: July 2002

To characterize and compare maxillary incisor lesions caused by various antitumor drugs, male BALB/c mice were given a single intravenous injection of an estimated 10% lethal dose (LD10) of 5-fluorouracil (5-FU), adriamycin (ADR), mitomycin C (MMC), vinblastine sulfate (VBL), taxotere (TXR), irinotecan hydrochloride CPT-11 , DX-8951f, or cisplatin (CDDP). After 3, 5, 10, 15, and 60 days, the animals were sacrificed, and the maxillary incisors were examined microscopically. The dental lesions observed were classified into 4 different types on the basis of their morphological features. The lesion due to 5-FU was characterized by focal defects in the dentin, and this injury was reversible (transient dentin injury). ADR- or MMC-induced lesions were defined by abnormal structure of the apical aspect of the tooth and irregular odontogenesis, lasting for a long period (persistent apical injury). Treatment with VBL or TXR showed irregular enamel formation and abnormal dentinogenesis. Their targets were considered to be both immature and mature odontogenic cells (diffuse dental injury). Exposure to CPT-11, DX-8951f, or CDDP elicited minor reductions in a few precursor cells in the epithelial sheath on day 3, but no prominent dental abnormalities were seen thereafter (nontoxic injury). In conclusion, antitumor drugs can cause a variety of dental lesions that vary temporally and spatially, making histopathological examination of the maxillary incisor an important component of the safety assessment process for novel antitumor drugs.
Morphological and Immunohistochemical Characteristics of the Heterogeneous Prostate-Like Glands (Paraurethral Gland) Seen in Female Brown-Norway Rats
Authors: Satoh Hiroshi , Mori Kazuhiko , Furuhama Kazuhisa
Published: July 2002

Morphological and immunohistochemical characteristics of the prostate-like glands (paraurethral gland) seen spontaneously in female Brown-Norway (BN) rats were investigated by gross, and light and electron microscopic examination. At 9- to 10-weeks-old, the paraurethral gland was detected in 50 out of 52 female animals examined (96.2%), and it was observed as single or paired structures located ventrolaterally in the urethra just caudal to neck of the urinary bladder. Microscopically, the glandular acini consisted of flat to cuboidal secretory epithelium surrounded by the smooth muscle. The glands displayed modest secreting activity, and a few secreting materials were observed in the acinar lumens. The main peripheral ducts were located in the urethral wall, and drained into the urethra on both sides. Ultrastructurally, abundant rough endoplasmic reticulum (RER), numerous mitochondria and lysosomes, and secretory granules in the apical portion of the epithelial cells were noted, and basal cells were also observed. These gland epithelial cells showed positive reactions when stained for androgen receptor (AR), prostate specific antigen (PSA), or prostate specific acid phosphatase (PSAP). The nature of this paraurethral gland resembled that of the prostate gland in male rats. Thus, the paraurethral glands seen in the females were considered homologous to the prostate gland in males.
A Survey of Discriminant Methods Used in Toxicological Histopathology
Authors: Holland Tom
Published: July 2002

A cluster survey of United Kingdom (UK)-based toxicological pathologists has been made of the methods used to discriminate treatment-related from spontaneous variation in histopathological material from toxicity studies. The response rate was 78% of 102 polled. Respondents overwhelmingly preferred methods based on grading or scoring lesions and comparatively avoided methods based on ranking, measuring, counting, pairwise comparisons, or overviewing. This preference was in spite of regarding parametric and ranking methods as both more powerful and specific than some commonly used scoring methods. Respondents were evenly divided on the use of blind examination and tended to avoid using historic material to form an opinion.
Species Differences in the Distribution of Drug-Metabolizing Enzymes in the Pancreas
Authors: Ulrich Alexis B. , Standop Jens , Schmied Bruno M. , Schneider Matthias B. , Lawson Terence A. , Pour Parviz M.
Published: July 2002

We investigated the cellular expression of 9 cytochrome P450-isozymes (CYP1A1, CYP1A2, CYP2B6, CYP2C8,9,19, CYP2D1, CYP2E1, CYP3A1, CYP3A2, CYP3A4) and 3 glutathione S-transferase-isozymes (GST- π , GST- α, GST- μ ) in the pancreas of hamsters, mice, rats, rabbits, pigs, dogs and monkeys, and in comparison with the human pancreas. A wide variation was found in the cellular localization of these enzymes between the 8 species. Most enzymes were expressed in the pancreas of the hamster, mouse, monkey and human, whereas rats, pigs, rabbits and dogs were lacking several isozymes. However, in all of the species the islet cells expressed more enzymes than ductal and acinar cells. An exclusive expression of enzymes in the islet cells was found in the hamster (CYP2E1), mouse (CYP1A1, CYP1A2, GST- α, GST- μ ), rat (CYP2C8,9,19), rabbit (CYP1A2, CYP2B6, GST- π), and pig (CYP1A1). Although no polymorphism was found in the pancreas of animals, in human tissue four enzymes were missing in about 50% of the cases. The results imply a greater importance of the islet cells in the metabolism of xenobiotics within the pancreas. The differences in the distribution of these drug-metabolizing enzymes in the pancreas between the species call for caution when extrapolating experimental results to humans.
Measuring the Digital Divide: A Framework for the Analysis of Cross-Country Differences
Authors: Corrocher Nicoletta , Ordanini Andrea
Published: July 2002

This article proposes a new model for measuring the digital divide within a set of countries or geographical areas. Starting from a series of elementary indicators the methodology groups these indicators into six factors of digitalization and, subsequently, aggregates the factors in a synthetic index called the synthetic index of digitalization. The dispersion in the distribution of the synthetic indexes of digitalization constitutes the measure of the digital divide. This method is based upon a measurement approach, which is different from the ones previously developed, since it uses principal components analysis for aggregating the variables and avoids many of the problems and limits shown by existing models. In the article an application of the methodology is provided within a set of ten developed countries for 2000 and 2001. The measurement framework for the digital divide presented here reveals new policy implications for public institutions and highlights opportunities and risks for managers working in the ‘digital economy’ environment.
Spin-Label Studies on Synaptic Membrane Lipids: Effect of Cholinergic Agonists, Antagonists, and Cholinesterase Inhibitors
Authors: Broomfield Clarence A. , Filbert Margaret G.
Published: July 2002

The effects on membrane fluidity of inhibitors of acetylcholinesterase (A ChE) as well as those of certain nicotinic agonists have been examined in synaptosomes prepared from the electroplax of Torpedo californica. Electron paramagnetic resonance (EPR) spectrometry was used to monitor changes in the order parameters of three spin-labeled fatty acid probes, doxyl stearic acid derivatives, intercalated in the lipid bilayer of synaptosomal membranes. Neither inhibition of A ChE nor the application of nicotinic ligands significantly influenced the viscosity of the membrane bilayer as judged by changes in the order parameters of the probes. A small, narrow line component of the EPR spectra disappeared when high concentrations of acetylcholine (ACh) were added to the synaptosomes. This effect was not seen with other cholinergic agonists, e.g., car-bachol. The results of this investigation suggest that if anti-A ChEs produce damaging effects on the lipid components of nerve membranes, the effects are indirect, resulting from accumulation of high ACh levels and not from a direct action of the agent on the membrane lipid.
A Role for Caspases in Lens Fiber Differentiation
Authors: Ishizaki Yasuki , Jacobson Michael D. , Raff Martin C.
Published: July 2002

There is increasing evidence that programmed cell death (PCD) depends on a novel family of intracellular cysteine proteases, called caspases, that includes the Ced-3 protease in the nematode Caenorhabditis elegans and the interleukin-1β–converting enzyme (ICE)-like proteases in mammals. Some developing cells, including lens epithelial cells, erythroblasts, and keratinocytes, lose their nucleus and other organelles when they terminally differentiate, but it is not known whether the enzymatic machinery of PCD is involved in any of these normal differentiation events. We show here that at least one CPP32 (caspase-3)-like member of the caspase family becomes activated when rodent lens epithelial cells terminally differentiate into anucleate lens fibers in vivo, and that a peptide inhibitor of these proteases blocks the denucleation process in an in vitro model of lens fiber differentiation. These findings suggest that at least part of the machinery of PCD is involved in lens fiber differentiation.
Integrin-regulated Secretion of Interleukin 4: A Novel Pathway of Mechanotransduction in Human Articular Chondrocytes
Authors: Millward-Sadler S.J. , Wright M.O. , Lee H.-S. , Nishida K. , Caldwell H. , Nuki G. , Salter D.M.
Published: July 2002

Chondrocyte function is regulated partly by mechanical stimulation. Optimal mechanical stimulation maintains articular cartilage integrity, whereas abnormal mechanical stimulation results in development and progression of osteoarthritis (OA). The responses of signal transduction pathways in human articular chondrocytes (HAC) to mechanical stimuli remain unclear. Previous work has shown the involvement of integrins and integrin-associated signaling pathways in activation of plasma membrane apamin-sensitive Ca2+-activated K+ channels that results in membrane hyperpolarization of HAC after 0.33 Hz cyclical mechanical stimulation. To further investigate mechanotransduction pathways in HAC and show that the hyperpolarization response to mechanical stimulation is a result of an integrin-dependent release of a transferable secreted factor, we used this response. Neutralizing antibodies to interleukin 4 (IL-4) and IL-4 receptor α inhibit mechanically induced membrane hyperpolarization and anti–IL-4 antibodies neutralize the hyperpolarizing activity of medium from mechanically stimulated cells. Antibodies to interleukin 1β (IL-1β) and cytokine receptors, interleukin 1 receptor type I and the common γ chain/CD132 (γ) have no effect on me- chanically induced membrane hyperpolarization. Chondrocytes from IL-4 knockout mice fail to show a membrane hyperpolarization response to cyclical mechanical stimulation. Mechanically induced release of the chondroprotective cytokine IL-4 from HAC with subsequent autocrine/paracrine activity is likely to be an important regulatory pathway in the maintenance of articular cartilage structure and function. Finally, dysfunction of this pathway may be implicated in OA.
Uncapping and Deregulation of Telomeres Lead to Detrimental Cellular Consequences in Yeast
Authors: Smith Christopher D. , Blackburn Elizabeth H.
Published: July 2002

Telomeres are the protein–nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen “monster” cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.
Mcm2, but Not Rpa, Is a Component of the Mammalian Early G1-Phase Prereplication Complex
Authors: Dimitrova Daniela S. , Todorov Ivan T. , Melendy Thomas , Gilbert David M.
Published: July 2002

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1–mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.
Primary Megakaryocytes Reveal a Role for Transcription Factor Nf-E2 in Integrin αiibβ3 Signaling
Authors: Shiraga Masamichi , Ritchie Alec , Aidoudi Sallouha , Baron Veronique , Wilcox David , White Gilbert , Ybarrondo Belen , Murphy George , Leavitt Andrew , Shattil Sanford
Published: July 2002

Platelet integrin αIIbβ3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of αIIbβ3 signaling remain poorly understood. To better understand this process, we established conditions to study αIIbβ3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of αIIbβ3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated β3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of αIIbβ3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2–deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of αIIbβ3 is controlled by NF-E2–regulated signaling pathways that mature late in megakaryocyte development and converge at the β3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional αIIbβ3 signaling.
Activity-Dependent Neuronal Control of Gap-Junctional Communication in Astrocytes
Authors: Rouach Nathalie , Glowinski Jacques , Giaume Christian
Published: July 2002

A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication.
Ablation of Uroplakin III Gene Results in Small Urothelial Plaques, Urothelial Leakage, and Vesicoureteral Reflux
Authors: Hu Ping , Deng Fang-Ming , Liang Feng-Xia , Hu Chuan-Min , Auerbach Anna B. , Shapiro Ellen , Wu Xue-Ru , Kachar Bechara , Sun Tung-Tien
Published: July 2002

Urothelium synthesizes a group of integral membrane proteins called uroplakins, which form two-dimensional crystals (urothelial plaques) covering >90% of the apical urothelial surface. We show that the ablation of the mouse uroplakin III (UPIII) gene leads to overexpression, defective glycosylation, and abnormal targeting of uroplakin Ib, the presumed partner of UPIII. The UPIII-depleted urothelium features small plaques, becomes leaky, and has enlarged ureteral orifices resulting in the back flow of urine, hydronephrosis, and altered renal function indicators. Thus, UPIII is an integral subunit of the urothelial plaque and contributes to the permeability barrier function of the urothelium, and UPIII deficiency can lead to global anomalies in the urinary tract. The ablation of a single urothelial-specific gene can therefore cause primary vesicoureteral reflux (VUR), a hereditary disease affecting ∼1% of pregnancies and representing a leading cause of renal failure in infants. The fact that VUR caused by UPIII deletion seems distinct from that caused by the deletion of angiotensin receptor II gene suggests the existence of VUR subtypes. Mutations in multiple gene, including some that are urothelial specific, may therefore cause different subtypes of primary reflux. Studies of VUR in animal models caused by well-defined genetic defects should lead to improved molecular classification, prenatal diagnosis, and therapy of this important hereditary problem.
Drosophila Aurora B Kinase Is Required for Histone H3 Phosphorylation and Condensin Recruitment during Chromosome Condensation and to Organize the Central Spindle during Cytokinesis
Authors: Giet Régis , Glover David M.
Published: July 2002

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95–105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103–1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.
Role of Diacylglycerol Kinase α in the Attenuation of Receptor Signaling
Authors: Sanjuán Miguel Angel , Jones David R. , Izquierdo Manuel , Mérida Isabel
Published: July 2002

Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGKα, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGKα-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH2-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid–mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGKα exerts on diacylglycerol-derived cell responses. This study is the first to describe DGKα as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.
A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic
Authors: Short Benjamin , Preisinger Christian , Körner Roman , Kopajtich Robert , Byron Olwyn , Barr Francis A.
Published: July 2002

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.
Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis
Authors: Conner Sean D. , Schmid Sandra L.
Published: July 2002

The μ2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that μ2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896–900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of α-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the μ subunit in vitro, and stage-specific assays for endocytosis show that μ phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous μ2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.
Claudin-based tight junctions are crucial for the mammalian epidermal barrier
Authors: Furuse Mikio , Hata Masaki , Furuse Kyoko , Yoshida Yoko , Haratake Akinori , Sugitani Yoshinobu , Noda Tetsuo , Kubo Akiharu , Tsukita Shoichiro
Published: July 2002

The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4–positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface, whereas in the claudin-1–deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.
14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport
Authors: Brunet Anne , Kanai Fumihiko , Stehn Justine , Xu Jian , Sarbassova Dilara , Frangioni John V. , Dalal Sorab N. , DeCaprio James A. , Greenberg Michael E. , Yaffe Michael B.
Published: July 2002

14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal α-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm.
Loss of ATP Diphosphohydrolase Activity with Endothelial Cell Activation
Authors: Robson Simon C. , Kaczmarek Elzbieta , Siegel Jonathan B. , Candinas Daniel , Koziak Katarzyna , Millan Maria , Hancock Wayne W. , Bach Fritz H.
Published: July 2002

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.
Light Chain Usage in Anti–double-stranded DNA B Cell Subsets: Role in Cell Fate Determination
Authors: Spatz Linda , Saenko Vladimir , Iliev Andrey , Jones Lori , Geskin Larisa , Diamond Betty
Published: July 2002

Two major mechanisms for the regulation of autoreactive B cells that arise in the bone marrow are functional silencing (anergy) and deletion. Studies to date suggest that low avidity interactions between B cells and autoantigen lead to B cell silencing, whereas high avidity interactions lead to deletion. Anti–double stranded (ds) DNA antibodies represent a pathogenic autospecificity in Systemic Lupus Erythematosus (SLE). An understanding of their regulation is critical to an understanding of SLE. We now demonstrate in a transgenic model in which mice express the heavy chain of a potentially pathogenic anti-DNA antibody that antibody affinity for dsDNA does not alone determine the fate of anti-dsDNA B cells. B cells making antibodies with similar affinities for dsDNA are regulated differently, depending on light chain usage. A major implication of this observation is that dsDNA may not be the self antigen responsible for cell fate determinations of anti-dsDNA B cells. Light chain usage may determine antigenic crossreactivity, and cross-reactive antigens may regulate B cells that also bind dsDNA.
Lipopolysaccharide (LPS)-induced Macrophage Activation and Signal Transduction in the Absence of Src-Family Kinases Hck, Fgr, and Lyn
Authors: Meng Fanying , Lowell Clifford A.
Published: July 2002

Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to generate null mutations of all three kinases in a single mouse strain. hck−/−fgr−/−lyn−/− mice are moderately healthy and fertile; macrophages cultured from these mice express normal levels of CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck−/−fgr−/−lyn−/− mice, functional analyses indicate that both elicited peritoneal (PEMs) and bone marrow–derived macrophages (BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-α) are normal or even enhanced in hck−/−fgr−/−lyn−/− macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription factor NF-κB, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not obligatory for LPS-initiated signal transduction.
Measles Virus Suppresses Cell-mediated Immunity by Interfering with the Survival and Functions of Dendritic and T Cells
Authors: Fugier-Vivier Isabelle , Servet-Delprat Christine , Rivailler Pierre , Rissoan Marie-Clotilde , Liu Yong-Jun , Rabourdin-Combe Chantal
Published: July 2002

Secondary infections due to a marked immunosuppression have long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. The mechanisms underlying the inhibition of cell-mediated immunity are not clearly understood but dysfunctions of monocytes as antigen-presenting cells (APC) are implicated. In this report, we demonstrate that measles virus (MV) replicates weakly in the resting dendritic cells (DC) as in lipopolysaccharide-activated monocytes, but intensively in CD40-activated DC. The interaction of MV-infected DC with T cells not only induces syncytia formation where MV undergoes massive replication, but also leads to an impairment of DC and T cell function and cell death. CD40-activated DC decrease their capacity to produce interleukin (IL) 12, and T cells are unable to proliferate in response to MV-infected DC stimulation. A massive apoptosis of both DC and T cells is observed in the MV pulsed DC–T cell cocultures. This study suggests that DC represent a major target of MV. The enhanced MV replication during DC–T cell interaction, leading to an IL-12 production decrease and the deletion of DC and T cells, may be the essential mechanism of immunosuppression induced by MV.
The Orphan Receptor CRF2-4 Is an Essential Subunit of the Interleukin 10 Receptor
Authors: Spencer Susan D. , Di Marco Francesco , Hooley Jeff , Pitts-Meek Sharon , Bauer Michele , Ryan Anne M. , Sordat Bernard , Gibbs Verna C. , Aguet Michel
Published: July 2002

The orphan receptor CRF2-4 is a member of the class II cytokine receptor family (CRF2), which includes the interferon receptors, the interleukin (IL) 10 receptor, and tissue factor. CRFB4, the gene encoding CRF2-4, is located within a gene cluster on human chromosome 21 that comprises three interferon receptor subunits. To elucidate the role of CRF2-4, we disrupted the CRFB4 gene in mice by means of homologous recombination. Mice lacking CRF2-4 show no overt abnormalities, grow normally, and are fertile. CRF2-4 deficient cells are normally responsive to type I and type II interferons, but lack responsiveness to IL-10. By ∼12 wk of age, the majority of mutant mice raised in a conventional facility developed a chronic colitis and splenomegaly. Thus, CRFB4 mutant mice recapitulate the phenotype of IL-10–deficient mice. These findings suggest that CRF2-4 is essential for IL-10–mediated effects and is a subunit of the IL-10 receptor.