bluuebunny/crossref_metadata_2025 huggingface dataset:
The use of coefficients to consider the incidences of hidden uncertainties
Authors: Bignoli A.J.
Published: October 2004

Linguistic qualifications of hidden uncertainties of literary variables appear at the beginning of the Structural Codes. These literary variables (like “care of designer”, “capacity of builder”, etc.) have influences on the numerical values of classical variables defined in the codes (like “resistance”, “areas of reinforcement”, “depth of sections”, “loads”, etc. An attempt is made in this paper to transform the subjectively linguistic qualifications of literary variables to numerical values assessed by calculus using fuzzy arithmetic and fuzzy logic. To reach this target all qualifications of linguistic variables are given using fuzzy numbers established in two scales and chosen by consensus of experts. These qualifications are transformed into coefficients by “filtering” using the filters also established by consensus of experts. For classical variables considered “pro‐safety” PS and “anti‐safety” AS two different types of the coefficients are considered and, as a consequence, two different types of filters are used. Also maximum values of AS (anti‐safety) and minimum values of PS (pro‐safety) of the coefficients are given. Considerations on sensitivity of the approach due to subjectivity are made.
An uncertainty model of structural reliability with imprecise parameters of probability distributions
Authors: Utkin L.V.
Published: October 2004

An approach to compute bounds for the structural reliability by imprecise parameters of the stress and strength probability distributions is proposed. The approach is based on using imprecise probability theory and takes into account different types of independence of the stress, strength and their parameters. It is shown that computation of the imprecise stress‐strength model can be reduced to solution of a number of linear programming problems. Special cases of the exponentially distributed stress and strength are considered. Various numerical examples illustrate the approach and show the impact of the independence conditions on imprecision of results.
Fuzzy information and imprecise probability
Authors: Viertl R. , Hareter D.
Published: October 2004

There are different kinds of uncertainty in connection with statistics and probability models. Besides the fuzziness of data also the imprecision of a‐priori distributions is an essential feature of stochastic models in applied mathematical work. The paper is on statistical analysis for fuzzy data, fuzzy densities and its application in Bayesian inference, including a gereralized probability concept, called fuzzy probability distributions.
Fuzzy randomness – a contribution to imprecise probability
Authors: Möller B.
Published: October 2004

The formal description of data uncertainty as fuzzy randomness combines randomness and fuzziness in an uncertain model. This generalized uncertainty model permits the simultaneous consideration of randomness, fuzziness and fuzzy randomness. Variables and functions with the property of fuzzy randomness are introduced. The formulation of the latter using α‐levels and fuzzy bunch parameters permits the application of fuzzy randomness in the numerical simulation of engineering problems. The finite element method is extended by the inclusion of fuzzy randomness to yield the fuzzy stochastic finite element method (FSFEM). Examples demonstrate the advantage of the chosen formal description in practical applications.
Severe Hypertrophy of the Base of the Tongue in Adults
Authors: Mamede Rui Celso Martins , De Mello‐Filho Francisco Veríssimo , Dantas Roberto Oliveira
Published: October 2004

The lymphoid follicles at the base of the tongue can be detected when examining the pharynx of adults, but the presence of large follicles, denoted “severe” hypertrophy of the base of the tongue (HBT) is rare. The objective of the present study was to identify severe HBT cases and their symptoms and to correlate them with the presence of pharyngolaryngeal signs and esophageal symptoms of gastroesophageal reflux (GER) in patients seen at a laryngology clinic. Severe HBT was considered to be present when the follicles prevented the view of the epiglottis or were massively distributed through the pharynx and larynx. Five cases of severe HBT were detected among 306 patients submitted to videolaryngoscopy over a period of 2 years, corresponding to 1.6% (5/306) of the total sample studied. However, this index markedly increases to 4% (4/101) among patients with pharyngolaryngeal signs of GER and reached 7.5% (4/53) among patients presenting GER symptoms such as heartburn, regurgitation, retrosternal burning feeling, and dysphagia. The complaints due to severe HBT were noisy respiration, hoarseness, throat clearing, dry cough, globus pharyngeus, and nasal voice. We conclude that the frequency of hypertrophied follicles is increased in the presence of signs and symptoms of GER and those HBT symptoms are confused with those of GER, except for nasal voice and noisy respiration.
Ernährungsverhalten von Grundschulkindern:
Authors: Eschenbeck Heike , Kohlmann Carl-Walter
Published: October 2004

Zusammenfassung. Untersucht wurde die Bedeutung der Einflussfaktoren Geschlechtszugehörigkeit und problemorientierte Stressbewältigung für das Ernährungsverhalten von Grundschulkindern. Dabei wurde das Ernährungsverhalten multimethodal mit Selbsteinschätzung, Verhaltensbeobachtung und Ernährungsprotokoll erhoben. 196 Kinder der Klassenstufen 3 und 4 im Alter zwischen 7 und 12 Jahren nahmen an der Studie teil. Die Ergebnisse zeigten deutliche Zusammenhänge sowohl zwischen der Geschlechtszugehörigkeit und den Gesundheitsverhaltensweisen als auch zwischen einer problemorientierten Stressbewältigung und gesundheitsbezogenem Verhalten: Mädchen zeigten verglichen mit Jungen das günstigere gesundheitsbezogene Verhalten. Eine problemorientierte Bewältigung ging mit einem gesünderen Verhalten einher. Dabei blieb dieser Zusammenhang auch nach Kontrolle potentieller Einflussparameter (z.B. Geschlecht, Klassenstufe, selbstberichtetes Gesundheitsverhalten) substantiell.
A global model of the coupled sulfur/oxidant chemistry in the troposphere: The sulfur cycle
Authors: Berglen Tore F. , Berntsen Terje K. , Isaksen Ivar S. A. , Sundet Jostein K.
Published: October 2004

A sulfur cycle chemistry scheme with dimethyl sulfide (DMS), SO2, sulfate, H2S, and methanesulfonic acid (MSA) is included in the OsloCTM2 model, and concentrations of sulfur are calculated interactively with the oxidant chemistry. This allows more consistent estimates of aqueous phase oxidation of SO2 to sulfate by O3, H2O2, and HO2NO2. The year 1996 is chosen as the standard, and a model run with 1996 meteorology and emissions is compared with 1996 observations. The results agree well with observations overall, although the model tends to overestimate SO2 and underestimate sulfate in Northern Hemisphere winter owing to an oxidation limitation. A global budget for 1996 quantifying the various processes is investigated. Our model results give a global lifetime (global burden) of 1 day (0.25 Tg(S)) and 3.8 days (0.5 3 Tg(S)) for SO2 and sulfate. Differences between the Southern Hemisphere, characterized by natural emissions and by loss of SO2 by O3 and H2O2 oxidation, and the Northern Hemisphere, characterized by anthropogenic emissions and by large loss by dry deposition, are revealed. Significant changes in sulfur emissions have occurred over the last decades with decrease in the Unites States and Europe and increase in Southeast Asia. U.S., European, and Chinese SO2 emissions have changed by −17.6%, −47.5%, and +93%, respectively. To study the impact of emission changes on the atmospheric composition, we have calculated distributions using the Global Emissions Inventory Activity (GEIA) 1985 inventory. The changes in sulfur emissions have significant changes on the sulfur concentrations and also some effect upon the oxidants. Increased emissions of NOx and hydrocarbons in China enhance O3, but increased sulfur inhibit the increase. The SO2 oxidation by OH, which can lead to formation of new sulfate particles, is given special attention. The model run using GEIA 1985 anthropogenic emission inventory is compared with other model studies.
A study of long‐term trends in mineral dust aerosol distributions in Asia using a general circulation model
Authors: Mukai Makiko , Nakajima Teruyuki , Takemura Toshihiko
Published: October 2004

Dust events have been observed in Japan with high frequency since 2000. On the other hand, the frequency of dust storms is said to have decreased in the desert regions of China since about the middle of the 1970s. This study simulates dust storms and transportation of mineral dust aerosols in the east Asia region from 1981 to 2001 using an aerosol transport model, Spectral Radiation‐Transport Model for Aerosol Species (SPRINTARS), implemented in the Center for Climate System Research/National Institute for Environmental Studies atmospheric global circulation model, in order to investigate the main factors that control a dust event and its long‐term variation. The model was forced to simulate a real atmospheric condition by a nudging technique using European Centre for Medium‐Range Weather Forecasts reanalysis data on wind velocities, temperature, specific humidity, soil wetness, and snow depth. From a comparison between the long‐term change in the dust emission and model parameters, it is found that the wind speed near the surface level had a significant influence on the dust emission, and snow is also an important factor in the early spring dust emission. The simulated results suggested that dust emissions from northeast China have a great impact on dust mass concentration in downwind regions, such as the cities of northeastern China, Korea, and Japan. When the frequency of dust events was high in Japan, a low‐pressure system tended to develop over the northeast China region that caused strong winds. From 2000 to 2001 the simulated dust emission flux decreased in the Taklimakan desert and the northwestern part of China, while it increased in the Gobi desert and the northeastern part of China. Consequently, dust particles seem to be transported more from the latter region by prevailing westerlies in the springtime to downwind areas as actually observed. In spite of the similarity, however, there is still a large disagreement between observed and simulated dust frequencies and concentrations. A more realistic land surface and uplift mechanism of dust particles should be modeled to improve the model simulation. Desertification of the northeastern China region may be another reason for this disagreement.
Ice nucleation in internally mixed ammonium sulfate/dicarboxylic acid particles
Authors: Wise Matthew E. , Garland Rebecca M. , Tolbert Margaret A.
Published: October 2004

Recent studies have shown that tropospheric sulfate aerosols commonly contain 50% or more by mass organic species. The influence of these organics on the chemical and physical properties of sulfate aerosols is not fully established. Using an aerosol flow tube technique, we have determined ice nucleation temperatures for particles composed of ammonium sulfate and mixtures of ammonium sulfate with a series of dicarboxylic acids. A calibration curve was developed to allow us to convert the freezing temperatures to a saturation ratio required for ice nucleation. At levels detectable by our experimental technique we find that the freezing temperatures and critical ice saturation ratios of each system were identical, for a given water activity of the solution, even though the solutions contained varying fractions of inorganic and organic components. Further experiments showed that the freezing behavior of pure dicarboxylic acid particles was identical to that of the other systems studied if the water activity was identical. Although the apparent freezing temperatures reported here are substantially warmer than those predicted by the water activity based nucleation theory of T. Koop et al., we find that solution water activity defined the freezing conditions for the systems studied here.
Wave scattering with UV multilevel partitioning method: 2. Three‐dimensional problem of nonpenetrable surface scattering
Authors: Tsang Leung , Li Qin , Xu Peng , Chen Dong , Jandhyala Vikram
Published: October 2004

A UV multilevel partitioning method (UV‐MLP) is developed to solve scalar wave three‐dimensional (3‐D) scattering problem. The method consists of setting up a table of transmitting and receiving block size and their separation using fast coarse‐coarse sampling. For a specific scattering problem with given geometry, the scattering structure is partitioned into multilevel blocks. By looking up the rank in the static problem, the impedance matrix for a given transmitting and receiving block is expressed into a product of U and V matrix. In this paper the method is illustrated by applying to a 3‐D scattering problem of random nonpenetrable rough surface. The cases of Dirichelt and Neumann boundary conditions are treated. Numerical simulation results are illustrated. For 65,536 boundary unknowns on a rough surface, and using a single processor of 2.66 GHz, it takes about 34 CPU min and 1.8 Gb of memory to compute the solution using conjugate gradient iterations and multilevel UV to accelerate the matrix‐column vector multiplication.
Buckland Park all‐sky interferometric meteor radar
Authors: Holdsworth David A. , Reid Iain M. , Cervera Manuel A.
Published: October 2004

A VHF all‐sky interferometric meteor radar system has been developed and installed at Buckland Park, South Australia. The radar is portable, allows a wide range of operating parameters, and can also be operated as a boundary layer radar. The analysis techniques have been developed using extensive simulations in an attempt to improve on standard techniques used by previous investigators. The results suggest that although pulse repetition frequencies (PRFs) around 2 kHz allow meteor velocity and deceleration estimation, PRFs around 500 Hz maximize count rate and improve the quality of meteor echo height estimates for this radar. Typical results are presented, indicating the radar obtains annual count rate variation of between 9000 and 14,000 height resolvable underdense meteors per day.
Source parameters of the Bhuj earthquake, India of January 26, 2001 from height and gravity changes
Authors: Chandrasekhar D. V. , Mishra D. C. , Singh B. , Vijayakumar V. , Bürgmann Roland
Published: October 2004

Height and gravity measurements observed along a profile across the epicentral area before and after the January 26, 2001, Mw 7.6 Bhuj earthquake show a maximum uplift of 1.57 ± 0.5 m and a corresponding gravity change of −393 ± 18 μGal. A best‐fit, single‐dislocation model inverted from the height‐changes using non‐linear optimization methods indicates that the high‐slip rupture was well contained in the aftershock zone and likely did not break to depths shallower than ∼10 km. Source parameters arrived in the present study agree well with those provided by seismic inversions and the distribution of aftershocks. Gravity data over the epicentral area are well modeled by the preferred model; however, a strong influence of shallow hydrological processes is inferred for three sites, two located on the Banni plains, whose mean gravity change ∼280 μGal suggests a total mass redistribution of as much as 2.9 Mt.
Field observation of fluid circulation patterns in a normal fault system
Authors: Fairley Jerry P. , Hinds Jennifer J.
Published: October 2004

Faults are often assumed to be either barriers or conduits for subsurface fluid flow, although they may act as both, depending on the hydraulic architecture of the fault and the direction of flow with respect to the fault plane. Here we use high‐resolution (5 × 5 m spacing) ground temperature measurements to track geothermal discharge in the step‐over region of an active échelon normal fault in southeast Oregon. Our analysis demonstrates that the fault acts as a combination conduit‐barrier system and reveals complex, 3‐dimensional circulation patterns in the area of the fault step‐over. Although complex flow circulation patterns are likely to be present in most fault‐controlled flow systems, they are generally neglected in conceptual and numerical models. Improved understanding of this aspect of subsurface fluid flow is essential for developing better models of fault hydrology.
Thin plate neotectonic models of the Australian plate
Authors: Burbidge D. R.
Published: October 2004

Thin plate finite element models of the neotectonic deformation of the Australian plate have been calculated in order to estimate the stress and strain rate within the plate, specifically concentrating on the Australian continent. The model includes plate‐bounding faults, an anelastic brittle‐ductile layered rheology and the option of laterally varying elevation and heat flow. The results of the models are compared to (1) the velocity of geodetic benchmarks on the Australian plate, (2) the spreading rate of the mid‐oceanic ridges along the Australian plate's margins, (3) the direction of the maximum horizontal principal stress, (4) the stress regime within the plate, and (5) the crustal thickness estimated from the depth to the base of Mohorovicic discontinuity's transition zone. A variety of models are tested with a wide range of input parameters. The model with the smallest misfit with observations predicts that the strain rate for most of the Australian continent is approximately 10−17 s−1. This model has a slightly lower strain rate in the central Australia and is higher off the northern coast of Australia than for the rest of the continent. Strain rates of this magnitude would be difficult to observe from geodetic or geologic data for most parts of Australia but would be enough to generate much of the seismicity that has been observed over the last century.
Vorticity flux through the Yucatan Channel and Loop Current variability in the Gulf of Mexico
Authors: Oey Lie‐Yauw
Published: October 2004

Recent observations (the CANEK Program [Candela et al., 2002]) suggest that potential vorticity (PV) flux anomaly (VFA) at Yucatan Channel may serve as a useful indicator of Loop Current variability, including Loop Current extension, retraction, and eddy shedding. Intuitively, anticyclonic VFA extends the Loop Current into the Gulf of Mexico and cyclonic VFA causes retraction or even shedding. However, this intuition is inconsistent with PV conservation. The problem is reexamined here by careful analyses of the relation between VFA and Loop Current variability using (1) the results of a 15‐year numerical simulation of shedding specified with simple forcing, and (2) CANEK and satellite observations. Both model and observations indicate that Loop Current eddy shedding or retraction tends to occur shortly (1∼2 months) after the influx of VFA at Yucatan has turned anticyclonic, and that these events are sometimes preceded by a more prolonged period of influx of cyclonic VFA. These findings suggest that contrary to intuition, influx of cyclonic VFA tends to extend the Loop Current into the Gulf, thus making the Loop Current more susceptible to retract or shed an eddy, and influx of anticyclonic VFA may then “trigger” retraction or eddy shedding. However, the Loop Current's behaviors are much more complex than can be prescribed by these simple rules. A much longer observational data set, coupled with more refined model experiments and sophisticated analyses, is required to further quantify the phenomenon.
Origin and role of water ice clouds in the Martian water cycle as inferred from a general circulation model
Authors: Montmessin F. , Forget F. , Rannou P. , Cabane M. , Haberle R. M.
Published: October 2004

In this paper, we present the results obtained by the general circulation model developed at the Laboratoire de Météorologie Dynamique which has been used to simulate the Martian hydrological cycle. Our model, which employs a simplified cloud scheme, reproduces the observed Martian water cycle with unprecedented agreement. The modeled seasonal evolution of cloudiness, which also compares well with data, is described in terms of the meteorological phenomena that control the Martian cloud distribution. Whereas cloud formation in the tropical region results from seasonal changes in the overturning circulation, Polar Hood clouds are mostly driven by variations of atmospheric wave activity. A sensitivity study allows us to quantify the effects of the transport of water ice clouds on the seasonal evolution of the water cycle. The residence time of cloud particles is long enough to allow cloud advection over great distances (typically thousands of kilometers). Despite the relatively low proportion of clouds (∼10%) in the total atmospheric inventory of water, their ability to be transported over large distances generally acts at the expense of the north polar cap and generates a water cycle globally wetter by a factor of 2 than a cycle produced by a model neglecting cloud transport. Around aphelion season, clouds modulate the north to south migration of water in a significant fashion and participate just as much as vapor in the cross‐equatorial transport of total water. Most of the year, atmospheric waves generate an equatorward motion of water ice clouds near the polar vortex boundaries, partially balancing the opposite poleward flux of water vapor. The combination of both effects delays the return of water to the north polar cap and allows water to build up in the Martian tropics.
Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)
Authors: DREMINA Elena S. , SHAROV Victor S. , KUMAR Keshava , ZAIDI Asma , MICHAELIS Elias K. , SCHÖNEICH Christian
Published: October 2004

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2Δ21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2Δ21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2Δ21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2Δ21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2Δ21 to SERCA. In contrast, Bcl-2Δ21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2Δ21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2Δ21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.
Protein arginine methylation during lytic adenovirus infection
Authors: KZHYSHKOWSKA Julia , KREMMER Elisabeth , HOFMANN Markus , WOLF Hans , DOBNER Thomas
Published: October 2004

Arginine methylation of proteins affects major processes in the cell, including transcriptional regulation, mRNA metabolism, signal transduction and protein sorting. Arginine methylation of Ad (adenovirus) E1B 55-kDa-associated protein E1B-AP5 was recently described by us [Kzhyshkowska, Schutt, Liss, Kremmer, Stauber, Wolf and Dobner (2001) Biochem. J. 358, 305–314]. In this first example of protein arginine methylation analysis in Ad-infected cells, we investigated methylation of the E1B-AP5 and the viral L4-100 kDa protein. We demonstrate that E1B-AP5 methylation is enhanced during the course of infection in a cell-type-specific manner. We also show that L4-100 kDa is efficiently methylated in Ad-infected cells. L4-100 kDa formed complex with methyltransferase in vivo during productive infection, and can be methylated by HRMT1L2 (human protein arginine methyltransferase 1) in vitro. Comparative analysis of E1B-AP5 and L4-100 kDa protein methylation in Ad-infected HeLa, MCF-7 and H1299 cells revealed that the profile of protein arginine methylation correlates with the efficiency of Ad proteins production. Our results suggest that protein arginine methylation is an important host-cell function required for efficient Ad replication.
Roles of USF, Ikaros and Sp proteins in the transcriptional regulation of the human reduced folate carrier B promoter
Authors: LIU Mingjun , WHETSTINE Johnathan R. , PAYTON Scott G. , GE Yubin , FLATLEY Robin M. , MATHERLY Larry H.
Published: October 2004

The hRFC (human reduced folate carrier) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to seven non-coding regions (A1, A2, A, B, C, D and E) and at least four promoters. For the hRFC-B basal promoter, regulation involves binding of Sp (specificity protein) transcription factors to a critical GC-box. By transiently transfecting HT1080 cells with 5′- and 3′-deletion constructs spanning 1057 bp of upstream sequence, a transcriptionally important region was localized to 158 bp flanking the transcriptional start sites. By gel shift and chromatin immunoprecipitation assays, USF (upstream stimulatory factor), Sp1 and Ikaros-related proteins were bound to consensus elements (one E-box, two GC-box and three Ikaros) within this region. The functional importance of these elements was confirmed by transient tranfections of HT1080 cells with hRFC-B reporter constructs in which they were mutated, and by co-transfections of Drosophila Mel-2 cells with wild-type hRFC-B promoter and expression constructs for USF1, USF2a, Sp1 and Ikaros 2 and 8. Both USF1 and Sp1 proteins transactivated the hRFC-B promoter. Sp1 combined with USF1 resulted in a synergistic transactivation. Identical results were obtained with USF2a. Ikaros 2 was a repressor of hRFC-B promoter activity whose effects were partly reversed by the dominant-negative Ikaros 8. In HT1080 cells, transfection with Ikaros 2 decreased endogenous hRFC-B transcripts, whereas USF1 and Sp1 increased transcript levels. Ikaros 2 also decreased reporter gene activity and levels of acetylated chromatin associated with the endogenous promoter. Collectively, these results identify transcriptionally important regions in the hRFC-B promoter that include multiple GC-box, Ikaros and E-box elements. Our results also suggest that co-operative interactions between transcription factors Sp1 and USF are essential for high-level hRFC-B transactivation and imply that these effects are modulated by the family of Ikaros proteins and by histone acetylation.
The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin
Authors: RIGATO Igino , PASCOLO Lorella , FERNETTI Cristina , OSTROW J. Donald , TIRIBELLI Claudio
Published: October 2004

Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1). To test this hypothesis directly, [3H]UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin–Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls. As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2. [3H]UCB uptake was measured in the presence of 60 μM human serum albumin at a free (unbound) concentration of UCB (BF) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5′-[β,γ-methylene]triphosphate). MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly. [3H]UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 μM MK571 and followed saturative kinetics with Km=10±3 nM (BF) and Vmax=100±13 pmol·min−1·(mg of protein)−1. UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1. Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity.
Opposing actions of cellular retinol-binding protein and alcohol dehydrogenase control the balance between retinol storage and degradation
Authors: MOLOTKOV Andrei , GHYSELINCK Norbert B. , CHAMBON Pierre , DUESTER Gregg
Published: October 2004

Vitamin A homoeostasis requires the gene encoding cellular retinol-binding protein-1 (Crbp1) which stimulates conversion of retinol into retinyl esters that serve as a storage form of vitamin A. The gene encoding alcohol dehydrogenase-1 (Adh1) greatly facilitates degradative metabolism of excess retinol into retinoic acid to protect against toxic effects of high dietary vitamin A. Crbp1−/−/Adh1−/− double mutant mice were generated to explore whether the stimulatory effect of CRBP1 on retinyl ester formation is due to limitation of retinol oxidation by ADH1, and whether ADH1 limits retinyl ester formation by opposing CRBP1. Compared with wild-type mice, liver retinyl ester levels were greatly reduced in Crbp1−/− mice, but Adh1−/− mice exhibited a significant increase in liver retinyl esters. Importantly, relatively normal liver retinyl ester levels were restored in Crbp1−/−/Adh1−/− mice. During vitamin A deficiency, the additional loss of Adh1 completely prevented the excessive loss of liver retinyl esters observed in Crbp1−/− mice for the first 5 weeks of deficiency and greatly minimized this loss for up to 13 weeks. Crbp1−/− mice also exhibited increased metabolism of a dose of retinol into retinoic acid, and this increased metabolism was not observed in Crbp1−/−/Adh1−/− mice. Our findings suggest that opposing actions of CRBP1 and ADH1 enable a large fraction of liver retinol to remain esterified due to CRBP1 action, while continuously allowing some retinol to be oxidized to retinoic acid by ADH1 for degradative retinoid turnover under any dietary vitamin A conditions.
Dual sensitivity of sarcoplasmic/endoplasmic Ca2+-ATPase to cytosolic and endoplasmic reticulum Ca2+ as a mechanism of modulating cytosolic Ca2+ oscillations
Authors: YANO Kojiro , PETERSEN Ole H. , TEPIKIN Alexei V.
Published: October 2004

The effects of ER (endoplasmic reticulum) Ca2+ on cytosolic Ca2+ oscillations in pancreatic acinar cells were investigated using mathematical models of the Ca2+ oscillations. We first examined the mathematical model of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) to reproduce the highly co-operative inhibitory effect of Ca2+ in the ER lumen on ER Ca2+ uptake in the acinar cells. The model predicts that luminal Ca2+ would most probably inhibit the conversion of the conformation state with luminal Ca2+-binding sites (E2) into the conformation state with cytoplasmic Ca2+-binding sites (E1). The SERCA model derived from this prediction showed dose–response relationships to cytosolic and luminal Ca2+ concentrations that were consistent with the experimental data from the acinar cells. According to a mathematical model of cytosolic Ca2+ oscillations based on the modified SERCA model, a small decrease in the concentration of endoplasmic reticulum Ca2+ (approx. 20% of the total) was sufficient to abolish the oscillations. When a single type of IP3R (IP3 receptor) was included in the model, store depletion decreased the spike frequency. However, the frequency became less sensitive to store depletion when we added another type of IP3R with higher sensitivity to the concentration of free Ca2+ in the cytosol. Bifurcation analysis of the mathematical model showed that the loss of Ca2+ from the ER lumen decreased the sensitivity of cytosolic Ca2+ oscillations to IP3 [Ins(1,4,5)P3]. The addition of a high-affinity IP3R did not alter this property, but significantly decreased the sensitivity of the spike frequency to IP3. Our mathematical model demonstrates how luminal Ca2+, through its effect on Ca2+ uptake, can control cytosolic Ca2+ oscillations.
Sp1 and Smad transcription factors co-operate to mediate TGF-β-dependent activation of amyloid-β precursor protein gene transcription
Authors: DOCAGNE Fabian , GABRIEL Cecilia , LEBEURRIER Nathalie , LESNÉ Sylvain , HOMMET Yannick , PLAWINSKI Laurent , MacKENZIE Eric T. , VIVIEN Denis
Published: October 2004

Abnormal deposition of Aβ (amyloid-β peptide) is one of the hallmarks of AD (Alzheimer's disease). This peptide results from the processing and cleavage of its precursor protein, APP (amyloid-β precursor protein). We have demonstrated previously that TGF-β (transforming growth factor-β), which is overexpressed in AD patients, is capable of enhancing the synthesis of APP by astrocytes by a transcriptional mechanism leading to the accumulation of Aβ. In the present study, we aimed at further characterization of the molecular mechanisms sustaining this TGF-β-dependent transcriptional activity. We report the following findings: first, TGF-β is capable of inducing the transcriptional activity of a reporter gene construct corresponding to the +54/+74 region of the APP promoter, named APPTRE (APP TGF-β-responsive element); secondly, although this effect is mediated by a transduction pathway involving Smad3 (signalling mother against decapentaplegic peptide 3) and Smad4, Smad2 or other Smads failed to induce the activity of APPTRE. We also observed that the APPTRE sequence not only responds to the Smad3 transcription factor, but also the Sp1 (signal protein 1) transcription factor co-operates with Smads to potentiate the TGF-β-dependent activation of APP. TGF-β signalling induces the formation of nuclear complexes composed of Sp1, Smad3 and Smad4. Overall, the present study gives new insights for a better understanding of the fine molecular mechanisms occurring at the transcriptional level and regulating TGF-β-dependent transcription. In the context of AD, our results provide additional evidence for a key role for TGF-β in the regulation of Aβ production.
Requirement of different mitochondrial targeting sequences of the yeast mitochondrial transcription factor Mtf1p when synthesized in alternative translation systems
Authors: BISWAS Tapan K. , GETZ Godfrey S.
Published: October 2004

Mitochondrial (mt) translocation of the nuclearly encoded mt transcription factor Mtf1p appears to occur independent of a cleavable presequence, mt receptor, mt membrane potential or ATP [Biswas and Getz (2002) J. Biol. Chem. 277, 45704–45714]. To understand further the import strategy of Mtf1p, we investigated the import of the wild-type and N-terminal-truncated Mtf1p mutants synthesized in two different in vitro translation systems. These Mtf1p derivatives were generated either in the RRL (rabbit reticulocyte lysate) or in the WGE (wheat germ extract) translation system. Under the in vitro import conditions, the RRL-synthesized full-length Mtf1p but not the N-terminal-truncated Mtf1p product was efficiently imported into mitochondria, suggesting that the N-terminal sequence is important for its import. On the other hand, when these Mtf1p products were generated in the WGE system, surprisingly, the N-terminal-truncated products, but not the full-length protein, were effectively translocated into mitochondria. Despite these differences between the translation systems, in both cases, import occurs at a low temperature and has no requirement for a trypsin-sensitive mt receptor, mt membrane potential or ATP hydrolysis. Together, these observations suggest that, in the presence of certain cytoplasmic factors (derived from either RRL or WGE), Mtf1p is capable of using alternative import signals present in different regions of the protein. This appears to be the first example of usage of different targeting sequences for the transport of a single mt protein into the mt matrix.
Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum
Authors: SPOONER Robert A. , WATSON Peter D. , MARSDEN Catherine J. , SMITH Daniel C. , MOORE Katherine A. H. , COOK Jonathon P. , LORD J. Michael , ROBERTS Lynne M.
Published: October 2004

Cells expressing ricin B chain within the secretory pathway are significantly more resistant to intoxication by ricin holotoxin but not to other cytotoxins that exploit similar endocytic routes to the cytosol. Furthermore, cells expressing the related B chain of abrin are protected against both incoming abrin and ricin. These phenotypes can be correlated with the abilities of the respective B chains to form disulphide-linked A–B holotoxins, since abrin B chain forms heterodimers with either abrin or ricin A chains, whereas ricin B chain forms heterodimers with ricin A chain only. In the ricin B-expressing cells, this newly made lectin disappears with biphasic kinetics comprising a retention phase followed by slow turnover and disposal after disengagement from calnexin cycle components. Interference with ricin cytotoxicity occurs during the early retention phase when ricin B chain is associated with PDI (protein disulphide-isomerase). The data show that retrotranslocation of incoming toxin is impeded by PDI-catalysed formation of heterodimers between endogenous B and A chains derived from reduced holotoxin, thus proving that reduction of ricin occurs in the endoplasmic reticulum. In contrast with other toxins, ricin does not appear to require either proteolytic cleavage or unfolding for PDI-catalysed reduction.
Copper homoeostasis inDrosophila melanogasterS2 cells
Authors: SOUTHON Adam , BURKE Richard , NORGATE Melanie , BATTERHAM Philip , CAMAKARIS James
Published: October 2004

Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms.
Stabilization of model membranes during drying by compatible solutes involved in the stress tolerance of plants and microorganisms
Authors: HINCHA Dirk K. , HAGEMANN Martin
Published: October 2004

Many organisms accumulate compatible solutes under environmental stress conditions. Cyanobacteria accumulate compatible solutes in response to increased external salinity, with tolerance increasing from Suc (sucrose) or trehalose to 2-O-(α-D-glucopyranosyl)-glycerol and glycinebetaine accumulating species. It is not clear how these different solutes influence salt tolerance. One possible explanation may be a differential ability of these solutes to stabilize membranes under stress conditions. We therefore performed drying experiments with liposomes in the presence of compatible solutes. Suc, trehalose and sorbitol protected liposomes from leakage of a soluble marker and from membrane fusion during drying and rehydration. 2-O-(α-D-glucopyranosyl)-glycerol was less effective and glycinebetaine showed hardly any effect. In combination with Suc, the latter two solutes showed improved protection. Lipid-phase transitions are known to contribute to solute leakage from liposomes. We determined phase transitions in dry membranes in the absence or presence of the solutes, using Fourier-transform infrared spectroscopy. The ability of the solutes to decrease the phase transition temperature corresponded closely to their ability to protect the liposomes against solute leakage. All solutes interacted with the phosphate in the lipid headgroups. The magnitude of the shift in the asymmetric P=O stretching vibration correlated closely with the lipid-phase transition temperature. This indicates that the degree of membrane protection afforded by the solutes is mainly determined by their ability to interact with the membrane lipids. However, this is not a determinant of cellular protection against salt stress, as the solutes show a reverse order when ranked with regard to protection against these stresses.
The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants
Authors: THOUVENOT Benoit , CHARPENTIER Bruno , BRANLANT Christiane
Published: October 2004

The Escherichia coli multi-promoter region of the gapA gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, gapA mRNAs are mainly initiated at the highly efficient gapA P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Eσ70 (holoenzyme associated with σ70) RNAP (RNA polymerase) at promoter gapA P1, we show that this promoter is an extended −10 promoter that needs a −35 sequence for activity. The −35 sequence compensates for the presence of a suboptimal −10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a −10 extension and a −35 sequence. It reveals a complex array of RNAP–DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in gapA P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the −12 A:T pair is opened. Strong contacts with RNAP occur both with the −35 sequence and the TG extension, so that the σ4 and σ2 domains may simultaneously contact the promoter DNA. RNAP–DNA interactions were also detected immediately downstream of the −35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter gapA P1 is a very efficient promoter sharing common properties with both extended −10 and non-extended −10 promoters.
Upstream stimulatory factors, USF1 and USF2, bind to the human haem oxygenase-1 proximal promoterin vivoand regulate its transcription
Authors: HOCK Thomas D. , NICK Harry S. , AGARWAL Anupam
Published: October 2004

The human HO-1 (haem oxygenase-1) gene encodes a microsomal enzyme responsible for the breakdown of haem, and is also cytoprotective in response to various cellular insults. HO-1 transcription is induced by a vast array of compounds including, but certainly not limited to, haem and heavy metals such as cadmium. In the present study, we show that upstream stimulatory factors, USF1 and USF2, ubiquitous proteins belonging to the basic helix–loop–helix-leucine zipper family of transcription factors, constitutively bind to the class B E-box located in the proximal promoter of the human HO-1 gene and are responsible for the enhancement of HO-1 gene transcription in human renal proximal tubular epithelial cells. Dimethylsulphate in vivo footprinting studies have identified three protected guanine residues in the E-box of the HO-1 proximal promoter. One of these guanine contact points is essential for USF binding, and when mutated mimics a deletion mutation of the entire E-box palindrome sequence encompassing all three guanine contact points. Binding of USF1 and USF2 to the HO-1 E-box was confirmed by chromatin immunoprecipitation and gel-shift assays. Furthermore, we show that overexpression of USF1 or USF2 enhances the basal expression of HO-1 and that expression of a USF dominant negative form reduces its expression. These results demonstrate for the first time that USF proteins bind to the human HO-1 promoter in vivo and are required for high-level expression of HO-1 by haem and cadmium in human renal epithelial cells.
Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue
Authors: SHAN Lu , MARTI Thomas , SOLLID Ludvig M. , GRAY Gary M. , KHOSLA Chaitan
Published: October 2004

Prolyl endopeptidases have potential for treating coeliac sprue, a disease of the intestine caused by proteolytically resistant peptides from proline-rich prolamins of wheat, barley and rye. We compared the properties of three similar bacterial prolyl endopeptidases, including the known enzymes from Flavobacterium meningosepticum (FM) and Sphingomonas capsulate (SC) and a novel enzyme from Myxococcus xanthus (MX). These enzymes were interrogated with reference chromogenic substrates, as well as two related gluten peptides (PQPQLPYPQPQLP and LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), believed to play a key role in coeliac sprue pathogenesis. In vitro and in vivo studies were conducted to evaluate the activity, specificity and acid/protease stability of the enzymes. All peptidases were relatively resistant to acid, pancreatic proteases and membrane peptidases of the small intestinal mucosa. Although their activities against reference substrates were similar, the enzymes exhibited substantial differences with respect to chain length and subsite specificity. SC hydrolysed PQPQLPYPQPQLP well, but had negligible activity against LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF. In contrast, the FM and MX peptidases cleaved both substrates, although the FM enzyme acted more rapidly on LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF than MX. Whereas the FM enzyme showed a preference for Pro–Gln bonds, SC cleaved both Pro–Gln and Pro–Tyr bonds with comparable efficiency, and MX had a modest preference for Pro–(Tyr/Phe) sites over Pro–Gln sites. While a more comprehensive understanding of sequence and chain-length specificity may be needed to assess the relative utility of alternative prolyl endopeptidases for treating coeliac sprue, our present work has illustrated the diverse nature of this class of enzymes from the standpoint of proteolysing complex substrates such as gluten.