bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production
Authors: Zhu Ping , Ding Jin , Zhou Jun , Dong Wei-Jia , Fan Chun-Mei , Chen Zhi-Nan
Published: June 2005

Monocytes/macrophages play an important role in rheumatoid arthritis (RA) pathogenesis. They can activate fibroblasts through many molecules, including IL-1 and tumor necrosis factor-alpha, but there have been very few reports on the role of CD147 in RA. In our study, the results of flow cytometry reveal that the mean fluorescence intensity (MFI) of CD147 expression on CD14+ monocytes of peripheral blood from RA patients was higher than that in normal control and ankylosing spondylitis (AS) patients. The MFI of CD147 expression on the CD14+ monocytes in RA synovial fluid was higher than that in RA peripheral blood. Immunohistochemical staining shows that CD147 expression in RA synovium correlated with matrix metalloproteinase (MMP)-1 expression. A double immunofluorescent assay shows that CD147 was expressed on CD68+ cells in RA synovium. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using a chemotaxis assay in vitro and it was found that the addition of anti-CD147 antibody or a CD147 antagonistic peptide significantly decreased the chemotactic index of the mononuclear cells. The role of CD147 in MMP production and cell invasion in vitro were studied through the co-culture of human CD14+ monocytes or monocytic line THP-1 cells and human fibroblasts, as well as by gel zymography and an invasion assay. Significantly elevated release and activation of MMP-9 and/or MMP-2 were seen in the co-culture of human monocytes/THP-1 cells and fibroblasts compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays was also observed in the co-cultured cells. The addition of CD147 antagonistic peptide had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture. Our study demonstrates that the increased expression of CD147 on monocytes/macrophages in RA may be responsible for elevated MMP secretion, cell invasion and CyPA-mediated cell migration into the joints, all of which may contribute to the cartilage and bone destruction of RA. These findings, together with a better understanding of CD147, CyPA and RA, will help in the development of innovative therapeutic interventions for RA.
Protective effect of vasoactive intestinal peptide on bone destruction in the collagen-induced arthritis model of rheumatoid arthritis
Authors: Juarranz Yasmina , Abad Catalina , Martinez Carmen , Arranz Alicia , Gutierrez-Cañas Irene , Rosignoli Florencia , Gomariz Rosa P , Leceta Javier
Published: June 2005

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, characterized by the presence of inflammatory synovitis accompanied by destruction of joint cartilage and bone. Treatment with vasoactive intestinal peptide (VIP) prevents experimental arthritis in animal models by downregulation of both autoimmune and inflammatory components of the disease. The aim of this study was to characterize the protective effect of VIP on bone erosion in collagen-induced arthritis (CIA) in mice. We have studied the expression of different mediators implicated in bone homeostasis, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), receptor activator of nuclear factor-κB (RANK), receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), IL-1, IL-4, IL-6, IL-10, IL-11 and IL-17. Circulating cytokine levels were assessed by ELISA and the local expression of mediators were determined by RT-PCR in mRNA extracts from joints. VIP treatment resulted in decreased levels of circulating IL-6, IL-1β and TNFα, and increased levels of IL-4 and IL-10. CIA-mice treated with VIP presented a decrease in mRNA expression of IL-17, IL-11 in the joints. The ratio of RANKL to OPG decreased drastically in the joint after VIP treatment, which correlated with an increase in levels of circulating OPG in CIA mice treated with VIP. In addition, VIP treatment decreased the expression of mRNA for RANK, iNOS and COX-2. To investigate the molecular mechanisms involved, we tested the activity of NFκB and AP-1, two transcriptional factors closely related to joint erosion, by EMSA in synovial cells from CIA mice. VIP treatment in vivo was able to affect the transcriptional activity of both factors. Our data indicate that VIP is a viable candidate for the development of treatments for RA.
Measured energy consumption and carbon emissions of air-conditioning in UK office buildings
Authors: Knight Ian , Dunn Gavin
Published: June 2005

With the EU Energy Performance in Buildings Directive imminent in 2006, attention is focusing on how to reduce carbon emissions from buildings in practice. This research was undertaken to try to establish some ‘real’ buildingbased figures for the energy performance of air-conditioning systems in UK office buildings. One of the major questions to be answered is whether chilled beam and ceiling systems really are more efficient than traditional air-conditioning systems when used in the real world.1,2 This paper presents findings of a two-year field monitoring study investigating the energy performance of airconditioning (AC) systems in UK office buildings. The findings shown in this paper include: 1) the cooling power demand for the various air-conditioning systems monitored; 2) comparison of national energy consumption and carbon emissions bench-marks to the monitored systems; 3) comparison of the energy consumption and carbon emissions of reverse-cycle AC systems with traditional heating-only systems. The results show that chilled ceiling systems typically consume significantly less energy than the other generic AC types in the heating and cooling of office buildings. However, all the generic AC system types studied appeared capable of meeting current ‘good practice’ standards for energy use in the UK.

Practical application: The work provides an insight into the energy performance achieved by real AC systems as operated in UK Offices. When the EC Energy Performance in Buildings Directive becomes law in January 2006 the practically achievable energy and emissions performance of building services will fall more sharply into focus for designers. It is hoped that this paper will provide information useful to both designers and operators of AC systems in the UK as they attempt to reduce the impact of AC systems on the environment.
Airtightness testing of very large buildings: a case study
Authors: Sharples S , Closs S , Chilengwe N
Published: June 2005

The airtightness of a building envelope impacts upon uncontrolled air leakage, associated ventilation energy losses and occupant thermal comfort. The scale of these problems in large buildings would be expected to increase in relation to the potential size of possible leakage areas. Recent changes to UK Building Regulations have required new, large, nondomestic buildings (/ 1000 m2 floor area) to meet a specified standard of airtightness. A building’s airtightness is conventionally assessed using a steady state fan pressurization technique. There was some discussion in the airtightness testing industry that the upper practical limit of large buildings capable of being tested might be around 5000 m2 due to technical issues relating to fan size, power requirements, transportation and noise. The feasibility of testing very large buildings (over 5000 m2) with a steady state fan technique has been questioned. This paper demonstrates that such testing is feasible by describing the development, calibration and application of a 2 m diameter fan pressurisation system. The fan was tested on the largest building in the world (floor area 57 440 m2) ever to have its airtightness determined by steady state fan pressurization.

Practical Application: The changes to Building Regualtions Part L2 that came into effect on 1st April 2002 raised the profile of the pressure testing of large buildings for airtightness. There were doubts raised concerning the feasibility of pressure testing very large buildings but the paper demonstrates that it is technically and practically possible.
Pressure transient control: Part I — criteria for transient analysis and control
Authors: Swaffield J A , Campbell D P , Gormley M
Published: June 2005

Pressure transient propagation is a wholly natural consequence of any change in operating conditions for a fluid carrying system. Rapid changes in flow conditions generate surge conditions that may result in system failure. The analysis of these phenomena has progressed over the past 100 years from empirical research aimed at the protection of large-scale pipeline and plant networks to the development of computing simulations to support system design. Pressure surge analysis is therefore a consideration in the design and operation of all fluid systems: the objective of such an analysis being the prediction, control and suppression of transients. This paper presents the background to the development of surge alleviation, from traditional pipeline protection to applications within building drainage and vent systems, thereby stressing that the system failure consequences of transient propagation are dependent on the particular system, independent of absolute surge pressure, and that system protection criteria may be developed that apply regardless of the system or the severity of the transient.

Practical application: The control of air pressure transients in building drainage systems has been limited due to the need to attenuate positive pressure propagation via an open termination at roof level - a poor solution as the transient will have affected all system trap seals before reaching the relief vent. The Positive Air Pressure Attenuator - an expandable bag that controls the rate of change of entrained airflow within the system - reduces the possibility of trapseal loss due to positive transient propagation. Potentially this is a major contribution to vent system design that could revolutionize 150 years of design methodology.
Experimental-based study of the aerodynamics of super-high-speed elevators
Authors: Bai Hong-lei , Shen Gongxin , So Albert
Published: June 2005

Elevators can be considered to be the most important building services system in a modern super-high-rise building. At the same time, the speed record has also been elevating with recent record of 1010 m/min. The design and construction of super-high-speed elevators are thus demanded. The primary experimental measurements for the aerodynamic characteristics of super-high-speed elevators were conducted using a physical model for simulation. The instantaneous speed of a falling elevator car at five different positions as well as the average pressure in front of and behind the car along the hoistway were measured while four scenaria with different shapes of car design were tested. Parameters associated with the hoistway, including the open ratio and the blockage ratio, varied in the experiments. Results were produced with respect to the car having different shapes and hoistway parameters. The nondimensional pressure differences for different shapes of car and hoistway parameters were analyzed so that several instructive conclusions were arrived at. Finally, a new concept of ‘tunnel effected shape drag’ was introduced based on these experiments and this effect clearly differentiates the aerodynamic performance of a car moving inside a hoistway from a race-car moving on a race-track.

Practical application: The speed record of elevators keeps increasing because building are getting taller. The major hurdles to the development of super-high-speed elevators are the aerodynamics. The experiments detailed in this article help us to understand ways to handle the problems and to design elevator cars with shapes that are more aerodynamically friendly. 1 Introduction
A value engineering analysis of timber windows
Authors: Asif M , Muneer T , Kubie J
Published: June 2005

Cost effectiveness of products and services has become very important in today’s highly competitive market. The customer needs a product that accomplishes its required functions with economy. Value engineering is a very interesting management technique that analyses alternative solutions and identifies the best possible option. The presented work analyses various designs of timber and aluminium clad timber windows on value energy parameters to determine their overall functional performance and cost effectiveness. Life-cycle costing of windows has been carried out over a 40-year life span taking into account all the major cost factors involved. Running cost, the dominant element in life-cycle cost of windows, has been calculated for four possible maintenance options. Results have indicated that an ‘air-filled triple-glazed aluminium-clad timber window’ is the value engineered choice amongst the range of window designs studied; it is the most cost effective when employed with gas and paint for space heating and maintenance purposes respectively.
Assessment of the potential of roof turbine ventilators for bathroom ventilation
Authors: Kuo I-shiow , Lai Chi-ming
Published: June 2005

This study set out to investigate the potential of installing a new common roof turbine ventilator on to an existing bathroom ventilation system which serves 14 bathrooms in the first dormitory block of Leader University in Taiwan, and evaluate the overall ventilation performance of this new combination. Field measurements of airflow in the ventilation ducts and low-speed wind tunnel experiments for this turbine ventilator were carried out. The results showed that the combination of the roof turbine ventilator and bathroom ventilation were successful in achieving sufficient air change rate in bathrooms, and this ventilation design alternative is now proposed as a method of improving the indoor air environment in bathrooms.
Assessment of side exhaust systems for residential kitchens in Taiwan
Authors: Lai Chi-ming
Published: June 2005

In response to concerns about the health of housewives who are regularly exposed to excessive quantities of air pollutants when cooking, a number of domestic documents have recommended solutions such as: ‘decreasing the installation height of the range hoods’ or ‘using desk-top ventilators’. However, few if any studies have addressed the issue of how to make improvements to the traditional Taiwanese style of range hood. This study investigated the design and experimental assessment of side exhaust systems in a full-scale model kitchen. Tests were carried out to understand how different exhaust configurations could affect the overall exhaust performance. It was found that single-slot, twin-slot, and fence-slot exhausts close to the pot rims performed well in air exhalation, and ensured the indoor air quality in the kitchen was maintained an acceptable level.

Practical application: That innovative side-sucking exhausts, installed closer to the pot rim in the residential kitchens, considered the pathway of the aerosol and made improvements to the traditional style of range hood. The proposed prototypes ensured that the indoor air quality in the kitchen was maintained at an acceptable level.
An improved use of cooling degree-days for analysing chiller energy consumption in buildings
Authors: Day A R
Published: June 2005

Cooling degree-days have not commonly been used in building energy analysis due largely to a lack of understanding of how they should be defined. The extra complexity of cooling systems, including the existence of latent loads, means that simplified energy analysis techniques have been deemed too crude to be meaningful. This paper presents a detailed analysis of measured cooling energy data from a large air-conditioned site in Perth, Western Australia, which builds on previous theoretical work to develop a robust definition of cooling degree-day base temperature. The results show that not only do cooling degree-days give results suggested by the theory, but that the use of cooling energy performance lines can be used for more sophisticated energy analysis.

Practical application: The energy performance of buildings is becoming increasingly important to define. Energy monitoring and analysis techniques using degree days have usually been confined to heating systems. This paper sets out how cooling degree-days can be used to assess the operational performance of a refrigeration plant, using real data to demonstrate the potential of the method.
A Multitasking General Executive for Compound Continuous Tasks
Authors: Salvucci Dario D.
Published: June 2005

As cognitive architectures move to account for increasingly complex real‐world tasks, one of the most pressing challenges involves understanding and modeling human multitasking. Although a number of existing models now perform multitasking in real‐world scenarios, these models typically employ customized executives that schedule tasks for the particular domain but do not generalize easily to other domains. This article outlines a general executive for the Adaptive Control of Thought–Rational (ACT–R) cognitive architecture that, given independent models of individual tasks, schedules and interleaves the models' behavior into integrated multitasking behavior. To demonstrate the power of the proposed approach, the article describes an application to the domain of driving, showing how the general executive can interleave component subtasks of the driving task (namely, control and monitoring) and interleave driving with in‐vehicle secondary tasks (radio tuning and phone dialing).
Rational Analyses of Information Foraging on the Web
Authors: Pirolli Peter
Published: June 2005

This article describes rational analyses and cognitive models of Web users developed within information foraging theory. This is done by following the rational analysis methodology of (a) characterizing the problems posed by the environment, (b) developing rational analyses of behavioral solutions to those problems, and (c) developing cognitive models that approach the realization of those solutions. Navigation choice is modeled as a random utility model that uses spreading activation mechanisms that link proximal cues (information scent) that occur in Web browsers to internal user goals. Web‐site leaving is modeled as an ongoing assessment by the Web user of the expected benefits of continuing at a Web site as opposed to going elsewhere. These cost–benefit assessments are also based on spreading activation models of information scent. Evaluations include a computational model of Web user behavior called Scent‐Based Navigation and Information Foraging in the ACT Architecture, and the Law of Surfing, which characterizes the empirical distribution of the length of paths of visitors at a Web site.
Human Symbol Manipulation Within an Integrated Cognitive Architecture
Authors: Anderson John R.
Published: June 2005

This article describes the Adaptive Control of Thought–Rational (ACT–R) cognitive architecture (Anderson et al., 2004; Anderson & Lebiere, 1998) and its detailed application to the learning of algebraic symbol manipulation. The theory is applied to modeling the data from a study by Qin, Anderson, Silk, Stenger, & Carter (2004) in which children learn to solve linear equations and perfect their skills over a 6‐day period. Functional MRI data show that: (a) a motor region tracks the output of equation solutions, (b) a prefrontal region tracks the retrieval of declarative information, (c) a parietal region tracks the transformation of mental representations of the equation, (d) an anterior cingulate region tracks the setting of goal information to control the information flow, and (e) a caudate region tracks the firing of productions in the ACT–R model. The article concludes with an architectural comparison of the competence children display in this task and the competence that monkeys have shown in tasks that require manipulations of sequences of elements.
Modeling Parallelization and Flexibility Improvements in Skill Acquisition: From Dual Tasks to Complex Dynamic Skills
Authors: Taatgen Niels
Published: June 2005

Emerging parallel processing and increased flexibility during the acquisition of cognitive skills form a combination that is hard to reconcile with rule‐based models that often produce brittle behavior. Rule‐based models can exhibit these properties by adhering to 2 principles: that the model gradually learns task‐specific rules from instructions and experience, and that bottom‐up processing is used whenever possible. In a model of learning perfect time‐sharing in dual tasks (Schumacher et al., 2001), speedup learning and bottom‐up activation of instructions can explain parallel behavior. In a model of a complex dynamic task (Carnegie Mellon University Aegis Simulation Program [CMU‐ASP], Anderson et al., 2004), parallel behavior is explained by the transition from serially organized instructions to rules that are activated by both top‐down (goal‐driven) and bottom‐up (perceptually driven) factors. Parallelism lets the model opportunistically reorder instructions, leading to the gradual emergence of new task strategies.
A Strategy‐Based Interpretation of Stroop
Authors: Lovett Marsha C.
Published: June 2005

Most accounts of the Stroop effect (Stroop, 1935) emphasize its negative aspect, namely, that in particular situations, processing of an irrelevant stimulus dimension interferes with participants' performance of the instructed task. In contrast, this paper emphasizes the fact that, even with that interference, participants actually can (and usually do) exert enough control to perform the instructed task. An Adaptive Control of Thought–Rational (ACT–R) model of the Stroop task interprets this as a kind of learned strategic control. Specifically, the concept of utility is applied to the two processes that compete in the Stroop task, and a utility‐learning mechanism serves to update the corresponding utility values according to experience and hence influence the competition. This model both accounts for various extant Stroop results and makes novel predictions about when people can reduce their susceptibility to Stroop interference. These predictions are tested in three experiments that involve a double‐response variant of the Stroop task.
An Activation‐Based Model of Sentence Processing as Skilled Memory Retrieval
Authors: Lewis Richard L. , Vasishth Shravan
Published: June 2005

We present a detailed process theory of the moment‐by‐moment working‐memory retrievals and associated control structure that subserve sentence comprehension. The theory is derived from the application of independently motivated principles of memory and cognitive skill to the specialized task of sentence parsing. The resulting theory construes sentence processing as a series of skilled associative memory retrievals modulated by similarity‐based interference and fluctuating activation. The cognitive principles are formalized in computational form in the Adaptive Control of Thought–Rational (ACT–R) architecture, and our process model is realized in ACT–R. We present the results of 6 sets of simulations: 5 simulation sets provide quantitative accounts of the effects of length and structural interference on both unambiguous and garden‐path structures. A final simulation set provides a graded taxonomy of double center embeddings ranging from relatively easy to extremely difficult. The explanation of center‐embedding difficulty is a novel one that derives from the model' complete reliance on discriminating retrieval cues in the absence of an explicit representation of serial order information. All fits were obtained with only 1 free scaling parameter fixed across the simulations; all other parameters were ACT–R defaults. The modeling results support the hypothesis that fluctuating activation and similarity‐based interference are the key factors shaping working memory in sentence processing. We contrast the theory and empirical predictions with several related accounts of sentence‐processing complexity.
Estrogen Negatively Regulates Epidermal Growth Factor (EGF)-Mediated Signal Transducer and Activator of Transcription 5 Signaling in Human EGF Family Receptor-Overexpressing Breast Cancer Cells
Authors: Boerner Julie L. , Gibson Matthew A. , Fox Emily M. , Posner Erika D. , Parsons Sarah J. , Silva Corinne M. , Shupnik Margaret A.
Published: June 2005

Breast cancer cell growth may be stimulated by 17β-estradiol (E2) or growth factors like epidermal growth factor (EGF). However, tumors typically depend on only one of these pathways and may overexpress either estrogen receptor (ER) or EGF receptor (EGFR) and related family members. Tumors overexpressing EGFR are more aggressive than those expressing ER. Intracellular mediators of these growth-stimulatory pathways are not completely defined, but one potential common mediator of EGF and E2 signaling is the transcription factor signal transducer and activator of transcription 5 (STAT5). To investigate the role of STAT5 in potential crosstalk between E2 and EGF, MDA-MB231 and SKBr3 breast cancer cells, which are ER-negative and overexpress human EGF family receptors, were used. Introduction of ERα and treatment with E2 decreased EGF-induced tyrosine phosphorylation of STAT5b, basal and EGF-induced STAT5-mediated transcription, and EGF-stimulated DNA synthesis in these cells. Suppressive effects of E2-ΕRα were specific for STAT5, as EGF stimulation of MAPK was unaffected. Deletion/mutation analysis of ERα demonstrated that the DNA-binding domain was insufficient, and that the ligand-binding domain was required for these responses. ERα transcriptional activity was not necessary for suppression of STAT5 activity. Overexpression of c-Src did not prevent suppression of STAT5 activity by E2 and ERα. However, ERα did prevent basal increases in STAT5 activity with overexpressed c-Src. In the context of human EGF receptor family overexpression, E2-ER opposes EGF signaling by regulating STAT5 activity. STAT5 may be a crucial point of signaling for both E2 and growth factors in breast cancer cells, allowing targeted therapy for many types of breast tumors.
Handis a direct target of Tinman and GATA factors duringDrosophilacardiogenesis and hematopoiesis
Authors: Han Zhe , Olson Eric N.
Published: June 2005

The existence of hemangioblasts, which serve as common progenitors for hematopoietic cells and cardioblasts, has suggested a molecular link between cardiogenesis and hematopoiesis in Drosophila. However, the molecular mediators that might link hematopoiesis and cardiogenesis remain unknown. Here, we show that the highly conserved basic helix-loop-helix (bHLH)transcription factor Hand is expressed in cardioblasts, pericardial nephrocytes and hematopoietic progenitors. The homeodomain protein Tinman and the GATA factors Pannier and Serpent directly activate Hand in these cell types through a minimal enhancer, which is necessary and sufficient to drive Hand expression in these different cell types. Hand is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes,and by Serpent in hematopoietic progenitors in the lymph gland. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, and indicate that the transcriptional pathways involved in development of the cardiovascular, excretory and hematopoietic systems may be more closely related than previously appreciated.
DNA double-strand breaks and homology search: inferences from a species with incomplete pairing and synapsis
Authors: Calvente Adela , Viera Alberto , Page Jesús , Parra M. Teresa , Gómez Rocío , Suja José A. , Rufas Julio S. , Santos Juan L.
Published: June 2005

The relationship between meiotic recombination events and different patterns of pairing and synapsis has been analysed in prophase I spermatocytes of the grasshopper Stethophyma grossum, which exhibit very unusual meiotic characteristics, namely (1) the three shortest bivalents achieve full synapsis and do not show chiasma localisation; (2) the remaining eight bivalents show restricted synapsis and proximal chiasma localisation, and (3) the X chromosome remains unsynapsed. We have studied by means of immunofluorescence the localisation of the phosphorylated histone H2AX (γ-H2AX), which marks the sites of double-strand breaks; the SMC3 cohesin subunit, which is thought to have a close relationship to the development of the axial element (a synaptonemal complex component); and the recombinase RAD51. We observed a marked nuclear polarization of both the maturation of SMC3 cohesin axis and the ulterior appearance of γ-H2AX and RAD51 foci, these being exclusively restricted to those chromosomal regions that first form cohesin axis stretches. This polarised distribution of recombination events is maintained throughout prophase I over those autosomal regions that are undergoing, or about to undergo, synapsis. We propose that the restricted distribution of recombination events along the chromosomal axes in the spermatocytes is responsible for the incomplete presynaptic homologous alignment and, hence, for the partial synaptonemal complex formation displayed by most bivalents.
The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium
Authors: Wassmer Thomas , Froissard Marine , Plattner Helmut , Kissmehl Roland , Cohen Jean
Published: June 2005

The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion.
Matrix cell adhesion activation by non-adhesion proteins
Authors: Koblinski Jennifer E. , Wu Michael , Demeler Borries , Jacob Karin , Kleinman Hynda K.
Published: June 2005

Cell adhesion to the extracellular matrix is important in many biological processes. Various ligands and cell surface receptors have been defined. In vitro cell adhesion to matrix proteins and to other `adhesion' proteins is generally measured on plastic culture substrates. We have found that the presence of low levels of adhesion proteins, e.g. fibronectin, together with high concentrations of non-adhesion proteins, e.g. osteonectin, can promote cell attachment on plastic culture dishes. This promotion of adhesion occurs even when the concentrations of fibronectin, collagen and other adhesive proteins are too low to support cell attachment alone. Other non-adhesive proteins that have similar activity in `triggering' the attachment of cells to low levels of adhesion molecules include bovine serum albumin (BSA) and cytochrome C. The non-adhesive protein must be added to the plate first, or together with the low amount of the adhesion protein, to `activate' cell attachment. Adding the adhesion protein fibronectin to the plate first, followed by osteonectin, resulted in no `activation' of attachment. The non-adhesive protein did not bind to the adhesive protein nor did it alter the level of adhesive protein binding to the substrate. The non-adhesive protein did, however, expose integrin-binding sites of the adhesive protein fibronectin. These data confirm and extend previous data by others demonstrating the role of non-adhesive proteins in regulating the conformation and cell adhesive activity of matrix adhesion proteins on plastic surfaces. Such findings might explain contradictions in the literature about the activity of `adhesive proteins'.
Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells
Authors: Fogg Vanessa C. , Liu Chia-Jen , Margolis Ben
Published: June 2005

The formation and maintenance of tight junctions is essential for the development of epithelial cell polarity. Recently, a number of conserved polarity-regulating proteins have been shown to localize to epithelial tight junctions, and to play a role in the regulation of tight junction formation. The Crumbs3/PALS1/PATJ protein complex localizes at epithelial tight junctions and interacts with the polarity-regulating protein complex of Par6/Par3/aPKC. Overexpression of Crumbs3 in MDCKII cells leads to a delay in tight junction formation in these cells, suggesting a role in the regulation of tight junction development. Here we report new evidence that Crumbs3 indeed plays an essential role in tight junction formation. Mammary MCF10A cells express little endogenous Crumbs3 and fail to form tight junctions when grown under standard tissue culture conditions. The staining pattern of ZO-1, a tight junction marker, is fragmented, and other tight junction markers show either fragmented junctional expression or diffuse cytoplasmic staining. Expression of exogenous Crumbs3 induces the formation of tight junction structures marked by smooth, continuous ZO-1 staining at apical cell-cell junctions. A number of other tight junction markers, including claudin-1 and occludin, are also recruited to these junctions. Analysis by transmission electron microscopy and measurements of the transepithelial electrical resistance confirm that these structures are functional tight junctions. Mutations in either the Crumbs3 PDZ binding motif or the putative FERM binding motif lead to defects in the ability of Crumbs3 to promote tight junction development. Our results suggest that Crumbs3 plays an important role in epithelial tight junction formation, and also provide the first known functional role for the mammalian Crumbs FERM binding domain.
The meiotic spindle of theDrosophilaoocyte: the role of Centrosomin and the central aster
Authors: Riparbelli Maria Giovanna , Callaini Giuliano
Published: June 2005

We provide here the first evidence that a distinct midzone is present in the Drosophila melanogaster female meiosis I spindle. This region has the ability to bind the Pavarotti kinesin-like (PAV-KLP) and Abnormal spindle (Asp) proteins, indicating a correct organization of the central spindle microtubules. We also identified the core component centrosomal protein centrosomin (CNN) at an unexpected site within the anaphase I spindle, indicating a role for CNN during the biogenesis of the female meiotic apparatus. However, there are no apparent defects in the midzone organization of cnn oocytes, whereas defects occur later when the central aster forms. The primary mutant phenotype of cnn oocytes is the failure to form a developed central microtubule organizing center (MTOC), although twin meiosis II spindles usually do form. Thus the central MTOC may not be essential for the formation of the inner poles of twin meiosis II spindles, as generally proposed, but it might be involved in maintaining their proper spacing. We discuss the proposal that, in the presence of a central MTOC, a chromatin-driven mechanism of spindle assembly like that described during meiosis I may control the morphogenesis of the twin meiosis II spindles.
Candida albicans hyphae have a Spitzenkörper that is distinct from the polarisome found in yeast and pseudohyphae
Authors: Crampin Helen , Finley Kenneth , Gerami-Nejad Maryam , Court Helen , Gale Cheryl , Berman Judith , Sudbery Peter
Published: June 2005

Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkörper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkörper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkörper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkörper and polarisome are distinct structures, that the polarisome and Spitzenkörper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae.
Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells
Authors: Giner Daniel , Ñeco Patricia , Francés María del Mar , López Inmaculada , Viniegra Salvador , Gutiérrez Luis M.
Published: June 2005

Transmitted light images showed an intricate and dynamic cytoplasmic structural network in cultured bovine chromaffin cells observed under high magnification. These structures were sensitive to chemicals altering F-actin-myosin and colocalised with peripheral F-actin, β-actin and myosin II. Interestingly, secretagogues induced a Ca2+-dependent, rapid (>10 second) and transitory (60-second cycle) disassembling of these cortical structures. The simultaneous formation of channel-like structures perpendicular to the plasmalemma conducting vesicles to the cell limits and open spaces devoid of F-actin in the cytoplasm were also observed. Vesicles moved using F-actin pathways and avoided diffusion in open, empty zones. These reorganisations representing F-actin transfer from the cortical barrier to the adjacent cytoplasmic area have been also confirmed by studying fluorescence changes in cells expressing GFP-β-actin. Thus, these data support the function of F-actin-myosin II network acting simultaneously as a barrier and carrier system during secretion, and that transmitted light images could be used as an alternative to fluorescence in the study of cytoskeleton dynamics in neuroendocrine cells.
PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage
Authors: Li Shaohua , Bordoy Randi , Stanchi Fabio , Moser Markus , Braun Attila , Kudlacek Oliver , Wewer Ulla M. , Yurchenco Peter D. , Fässler Reinhard
Published: June 2005

PINCH1 is composed of 5 LIM domains, binds integrin-linked kinase (ILK) and locates to integrin-mediated adhesion sites. In order to investigate PINCH1 function we generated mice and embryonic stem (ES) cell-derived embryoid bodies (EBs) lacking the PINCH1 gene. Similar to mice lacking β1 integrin or Ilk, loss of PINCH1 arrested development at the peri-implantation stage. In contrast to β1 integrin or Ilk mutants, however, disruption of the PINCH1 gene produced implantation chambers with visible cell clumps even at embryonic day 9.5. In order to define the phenotype leading to the peri-implantation lethality we made PINCH1-null EBs and found similar but also additional defects not observed in β1 integrin or Ilk mutant EBs. The similarities included abnormal epiblast polarity, impaired cavitation and detachment of endoderm and epiblast from basement membranes. Additional defects, which were not observed in β1 integrin- or ILK-deficient mice or EBs, included abnormal cell-cell adhesion of endoderm and epiblast as well as the presence of apoptotic cells in the endodermal cell layer. Although ILK and PINCH1 were shown to be involved in the phosphorylation of serine-473 of PKB/Akt, immunostaining with specific antibodies revealed no apparent alteration of PKB/Akt phosphorylation in PINCH1-deficient EBs. Altogether these data demonstrate an important role of PINCH1 for integrin function, actin organization, cell-cell adhesion and endodermal cell survival during the implanting of mouse embryos.
PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa
Authors: Etournay Raphaël , El-Amraoui Aziz , Bahloul Amel , Blanchard Stéphane , Roux Isabelle , Pézeron Guillaume , Michalski Nicolas , Daviet Laurent , Hardelin Jean-Pierre , Legrain Pierre , Petit Christine
Published: June 2005

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.
A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts
Authors: Destaing Olivier , Saltel Frédéric , Gilquin Benoit , Chabadel Anne , Khochbin Saadi , Ory Stéphane , Jurdic Pierre
Published: June 2005

Osteoclast maturation is accompanied by changes in podosome patterning, resulting in the formation of a peripheral belt, which requires an intact microtubule network. Here, we report that by inhibiting Rho, the podosome belt is maintained at the cell periphery despite depolymerisation of microtubules by nocodazole. Rho inhibition was correlated to the increase in microtubule stabilisation and microtubule acetylation. By microinjecting activated Rho or its activated effector mDia2 in osteoclasts, we found that the podosome belt was disrupted and the level of microtubule acetylation dramatically decreased. We further characterised the molecular mechanism responsible for microtubule deacetylation by co-immunoprecipitation experiments. We found that not only was mDia2 coprecipitating with the recently identified microtubule deacetylase HDAC6 but that it also activated the microtubule deacetylase activity of HDAC6 in an in vitro deacetylase assay. Finally, we found that during osteoclastogenesis, there is a correlation between the increase in microtubule acetylation and the podosome belt stabilisation and that if Rho is inhibited in the early stages of osteoclast differentiation, it accelerates both microtubule acetylation and podosome belt stabilisation. Altogether, our data reveal a pathway in which Rho interferes with the osteoclast maturation process by controlling the level of microtubule acetylation and actin organisation through mDIA2 and HDAC6.
Glucocorticoids remodel nuclear envelope structure and permeability
Authors: Shahin Victor , Ludwig Yvonne , Schafer Claudia , Nikova Dessy , Oberleithner Hans
Published: June 2005

The present study describes glucocorticoid induced remodelling of nuclear envelope (NE) structure and permeability. A glucocorticoid analogue, triamcinolone acetonide (TA), is injected into Xenopus laevis oocytes that express an exogeneous glucocorticoid receptor (GR). Electrical, fluorescence and nano-imaging techniques are applied to study the permeability and the structure of the NE at 5 and 60 minutes after injection of TA. A remarkable dilation of nuclear pore complexes (NPCs), a rearrangement of NPC distribution and a significant increase of NE permeability for ions and fluorescent 20 kDa dextran are observed within 5 minutes of TA exposure. At regular distances on local NE patches, NPCs seem to adjoin forming clusters each consisting of several hundred NPCs. Interestingly, at the same time of exposure, hydrophobicity of NPC central channels and NPC-free NE surface increases. The changes in permeability and structure are transient as the NE permeability returns to its initial state within 60 minutes. In conclusion, the NE is a barrier of high plasticity sensitive to hydrophobic molecules. Remodelling of NE structure and permeability is a prerequisite for mediating physiological actions of glucocorticoids.
The C-terminus of prestin influences nonlinear capacitance and plasma membrane targeting
Authors: Zheng Jing , Du Guo-Guang , Matsuda Keiji , Orem Alex , Aguiñaga Sal , Deák Levente , Navarrete Enrique , Madison Laird D. , Dallos Peter
Published: June 2005

Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cell's membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G/Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestin's function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function.