bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative roles of rifting tectonics and magma ascent processes: Inferences from geophysical, structural, volcanological, and geochemical data for the Neapolitan volcanic region (southern Italy)
Authors: Piochi Monica , Bruno Pier Paolo , De Astis Gianfilippo
Published: July 2005

The Neapolitan volcanic region is located within the graben structure of the Campanian Plain (CP), which developed between the western sector of the Appenine Chain and the eastern margin of the Tyrrhenian Sea. Two volcanic areas, spaced less than 10 km apart, are situated within the CP: the Somma‐Vesuvius Volcano (SVV) and the Phlegraean Volcanic District (PVD). SVV is a typical stratovolcano, whereas PVD, including Campi Flegrei, Procida, and Ischia, is composed mostly of monogenetic centers. This contrast is due to different magma supply systems: a widespread fissure‐type system beneath the PVD and a central‐type magma supply system for the SVV. Volcanological, geophysical, and geochemical data show that magma viscosity, magma supply rate, and depth of magma storage are comparable at PVD and SVV, whereas different structural arrangements characterize the two areas. On the basis of geophysical data and magma geochemistry, an oblique‐extensional tectonic regime is proposed within the PVD, whereas in the SVV area a compressive stress regime dominates over extension. Geophysical data suggest that the area with the maximum deformation rate extends between the EW‐running 41st parallel and the NE‐running Magnaghi‐Sebeto fault systems. The PVD extensional area is a consequence of the Tyrrhenian Sea opening and is decoupled from the surrounding areas (Roccamonfina and Somma‐Vesuvius) which are still dominated by Adriatic slab dynamics. Spatially, we argue that the contribution of the asthenospheric wedge become much less important from W‐NW to E‐SE in the CP. The development of the two styles of volcanism in the CP reflects the different tectonic regimes acting in the area.
Study of the proton arc spreading effect on primary ionization rates
Authors: Fang Xiaohua , Liemohn Michael W. , Kozyra Janet U. , Solomon Stanley C.
Published: July 2005

We investigate the influence of the spatial spreading effect in a proton arc with finite width on resulting primary ionization rates, using our three‐dimensional (3‐D) Monte Carlo ion transport model. By the direct impact from energetic protons and generated hydrogen atoms, ambient neutrals in the Earth's upper atmosphere can be ionized in charge exchange or ionization collisions. The model results show that the ionization rates (and particle fluxes) depend on all of the parameters we varied: incident proton arc dimensions, energy spectra, average energies, latitudinal energy flux distributions, and magnetic field dip angles. It is found that a correction factor, often introduced at an equilibrium altitude (∼300 km) in one‐dimensional (1‐D) theoretical models, cannot completely account for the beam spreading effect for an incident proton arc of finite width. Below ∼300 km, ionization rates in 1‐D models are generally overestimated at high altitudes (above ∼150 km) and underestimated at low altitudes (below ∼150 km). The overestimation is caused by overlooking the difference between the spatial spreading for the particle fluxes and for the ionization rates. At low altitudes, the beam radius gets smaller, causing underestimation in the 1‐D ionization rates. The results of our 3‐D sensitivity study of various parameters can be applied in future studies of auroral and ring current proton precipitation into the upper atmosphere.
Experimental epithermal alteration of synthetic Los Angeles meteorite: Implications for the origin of Martian soils and identification of hydrothermal sites on Mars
Authors: Hurowitz Joel A. , McLennan Scott M. , Lindsley Donald H. , Schoonen Martin A. A.
Published: July 2005

The dissolution behavior and secondary minerals generated by experimental alteration of a synthetic Los Angeles basaltic shergottite (LA) were evaluated at epithermal (75°C) conditions. Starting pH values of 1.1 and 3.6 and water‐rock ratios ranging from 100 to 2000 were studied using flow‐through and batch reactor experimental apparatus. Dissolution of plagioclase dominates effluent solution chemistry under all experimental conditions. Little Mg is released to solution because nearly 100% of the Mg in unaltered LA is contained in pyroxene M1 sites, which are relatively resistant to dissolution. Secondary minerals generated include siliceous residue, amorphous SiO2, lepidocrocite and ferrihydrite, anhydrite, alunogen, rhomboclase, ferrinatrite, halite, and unidentified Ca and Na sulfates. The dominance of alteration products of plagioclase suggests that LA‐type basalts have not contributed significantly to the Martian soils. The search for hydrothermal sites by remote sensing may be hampered by the general similarity of the alteration products found in the near‐surface epithermal environment to those thought to exist in Martian soils (sulfates, iron oxyhydroxides, amorphous silica). However, the unique chemical composition of secondary products formed by LA alteration may make detection of recent hydrothermal alteration of evolved basaltic rocks more straightforward. In addition, alteration in terrestrial epithermal environments associated with mafic to intermediate volcanic rocks generally produces abundant sulfides and kaolinite, two potentially unique mineral phases which might also indicate the presence of active or extinct hydrothermal systems.
A new model for rain scatter interference for coordination between Earth stations and terrestrial stations
Authors: Gibbins Chris J.
Published: July 2005

Scattering from rain is known to be a source of possible interference between communications systems sharing the same frequency bands. A new model has been developed for estimating the transmission loss due to bistatic rain scatter between an Earth station and a terrestrial station. This model has particular application in the process of coordination of Earth stations with terrestrial stations operating in the same frequency bands, whereby detailed interference studies are carried out only for stations within an area beyond which harmful interference may be considered negligible. The new model includes the Earth station elevation angle as an input parameter, together with the most recent information on rainfall rates and surface water vapor densities included in the recommendations of the International Telecommunications Union (ITU), and is more flexible yet neutral in its overall impact when applied to the process of coordination as defined in Appendix 7 of the ITU Radio Regulations.
Sensitivity of microwave brightness temperatures to hydrometeors in a tropical deep convective cloud system at 89–190 GHz
Authors: Hong Gang , Heygster Georg , Miao Jungang , Kunzi Klaus
Published: July 2005

The sensitivity of microwave brightness temperatures at the frequencies between 89 and 190 GHz to surface emissivities and hydrometeors in a tropical deep convective cloud system is investigated by simulations, using the Goddard Cumulus Ensemble model data of a simulated oceanic tropical squall line as input for a radiative transfer model. It is found that only the window channel at 89 GHz has an apparent dependence on the surface emissivity. The three water vapor channels around 183 GHz (i.e., 183.3 ± 1, ±3, and ±7 GHz) are completely independent, and the window channel at 150 GHz is nearly independent of the surface emissivity because of the atmospheric opacity at these frequencies. All channels are apparently influenced by deep convective clouds and their outflowing thick cirrus clouds. The channels at 89, 150, and 183.3 ± 7 GHz are strongly sensitive to variations in the liquid water content at levels above 5 km. The sensitivity of the channel at 150 GHz to liquid water is about twice that at 183.3 ± 7 GHz. All channels are generally sensitive to variations in the frozen hydrometeor content at levels above 7 km. The 183.3 ± 1 GHz channel has virtually no influence from the frozen hydrometeors at levels below 7 km. The sensitivity suggests that it should be possible to estimate the frozen hydrometeor properties in levels above 7 km in tropical deep convective clouds using the water vapor channels around 183 GHz.
Late Orogenic doming in the eastern Betic Cordilleras: Final exhumation of the Nevado‐Filabride complex and its relation to basin genesis
Authors: Augier Romain , Jolivet Laurent , Robin Cécile
Published: July 2005

The geometry, timing, and kinematics of late orogenic extension in the Betic Cordilleras pose the problem of a decoupling of upper crustal and lower crustal deformation regimes. Perpendicular directions of extension in metamorphic domes and nearby sedimentary basins remain unexplained. This paper puts kinematic constraints on the final exhumation of the Nevado‐Filabride complex, focusing on the formation of metamorphic domes and their relations with the adjacent basins. Structural fabrics and kinematic indicators below the main shear zones as well as their relations with both published changing metamorphic P‐T conditions and geochronological data were studied. Our approach describes (1) a consistent top‐to‐the‐west shear parallel to dome axes of during D2 (i.e., during decompression) with distributed ductile flow and the onset of strain localization along major shear zones, (2) further strain localization along the major shear zones under greenschist facies conditions, during D3 leading to S‐C′ mylonites formation accompanied with a rock strong thickness reduction, (3) the divergence of shear direction on either limbs of domes during D3 showing the appearance of the dome geometry, and (4) a local evolution toward N‐S brittle extension (D4) in the upper plate and formation of sedimentary basins. Continuous ductile to brittle top‐to‐the‐west shear is compatible with the slab retreat hypothesis from the Miocene; the formation of domes which adds gravitational forces responsible for the final stages of exhumation is thus characterized by important kinematics changes necessary to explain coeval N‐S opened basins. Later, from the upper Tortonian, a contractional event (D5) amplified the earlier domal structures forming the present north vergent folds.
Water trading at the margin: The evolution of water markets in the Murray‐Darling Basin
Authors: Turral H. N. , Etchells T. , Malano H. M. M. , Wijedasa H. A. , Taylor P. , McMahon T. A. M. , Austin N.
Published: July 2005

Water trading in Australia is enabled by much historical institutional development, which had other objectives at the time that it was implemented. After 2 decades of institutional reform to enable water markets in the Murray Darling Basin, active markets are reallocating surface water entitlements among irrigation users. However, permanent water trading is currently limited in terms of the volume traded and reallocation among uses. Given these limitations, this paper seeks to assess the success of surface water markets in the Murray‐Darling Basin by comparing current practice against the six desirable characteristics for water markets suggested by Howe et al. (1986). Overall, it is argued that, despite the relatively low rate of reallocation, the market performs well against most criteria but that ongoing evolution of institutional arrangements is critical for improved success.
Role of geomorphic and hydraulic parameters in governing pore water seepage from salt marsh sediments
Authors: Gardner Leonard Robert
Published: July 2005

A numerical boundary integral equation model has been used to study the effects of marsh geomorphology and hydraulic parameters on the seepage of pore water into tidal channels and its subsequent recharge by tidal inundation. In general, the results show that regardless of the geomorphic configuration the locus of maximum seepage flux occurs at or near the intersection of the creek bank and the channel water surface. Over a tidal cycle, typically two thirds of the total seepage discharge occurs through the creek bank with only about a third discharging from the channel bottom. In most cases the volume of seepage discharging through the creek bank exceeds by a factor of two or more that emanating from the channel bottom. Of the creek bank discharge, up to half may occur through the seepage face that develops above the tide line at tidal stages below mean tide. The ratio of creek bank to bottom seepage only approaches unity when the thickness of the aquifer beneath the bottom is similar to or greater than the width of the channel bottom and the ratio of conductivity to specific yield exceeds 0.5. For given hydraulic parameters, total seepage volume varies directly with creek bank slope and aquifer thickness, whereas these factors are inversely related to the ratio of the two seepage components. Channel bottom width, on the other hand, has no influence on total seepage volume but greatly increases the component ratio as the width approaches zero. Since most of the seepage discharge is derived from sediments within several meters of the creek bank, variations in seepage discharge driven by variations in marsh geomorphology might play a role in the productivity of creekside cord grass. Several hypotheses are offered and discussed regarding the effects of geomorphology on creekside productivity. These results also indicate that placement of seepage meters only on the channel bottom will not give samples or measures representative of the total seepage.
The areal reduction factor: A multifractal analysis
Authors: Veneziano Daniele , Langousis Andreas
Published: July 2005

The areal reduction factor (ARF) ? is a key quantity in the design against hydrologic extremes. For a basin of area a and a duration d, ?(a, d, T) is the ratio between the average rainfall intensity in a and d with return period T and the average rainfall intensity at a point for the same d and T. Empirical ARF charts often display scaling behavior. For example, for large (/d) ratios and given T the ARF tends to behave like (/d)-a for some a. Here we obtain scaling properties of the ARF under the condition that space‐time rainfall has multifractal scale invariance. The scaling exponents of the ARF are related in a simple way to the multifractal properties of the parent rainfall process. We consider regular and highly elongated basins, quantify the effect of rainfall advection, and investigate the bias from estimating the ARF using sparse rain gauge networks. We also study the effects of departure of rainfall from exact multifractality. The results explain many features of empirical ARF charts, while suggesting dependencies on advection, basin shape, and return period that are difficult to quantify empirically. The theoretical scaling relations may be used to extrapolate the ARF beyond the empirical range of a, d, and T.
Wet subduction versus cold subduction
Authors: Litasov Konstantin D. , Ohtani Eiji , Sano Asami , Suzuki Akio , Funakoshi Kenichi
Published: July 2005

In situ X‐ray diffraction study of post‐spinel transformation in hydrous peridotite (2 wt.% H2O) indicates that the phase boundary is shifted to higher pressures by 0.6 GPa relative to anhydrous peridotite at 1473 K, whereas, it shows no obvious shift at high temperature around 1873 K. A linear equation for the boundary is P (GPa) = −0.002 T (K) + 26.3, which is applicable for temperatures below 1800 K. The present data shows that a significant part of the depressions seen in the 660‐km seismic discontinuity may be affected by existence of water. It is also well resolved that the olivine‐wadsleyite phase transformation corresponding to the 410‐km seismic discontinuity is shifted to lower pressures by 1–2 GPa by the addition of water. Thus, the topography of seismic discontinuities in the transition zone associated with slabs can be attributed not only to cold subduction but also wet subduction.
The simplest ENSO recharge oscillator
Authors: Burgers Gerrit , Jin Fei‐Fei , van Oldenborgh Geert Jan
Published: July 2005

Eastern Pacific sea surface temperature (SST) and mean equatorial Pacific thermocline depth are key variables in El Niño–Southern Oscillation (ENSO). A linear fit to observations leads to a remarkably simple picture: ENSO can be represented by a classical damped oscillator, with SST and thermocline depth playing the roles of momentum and position, respectively. An independent fit of observed relationships between western and eastern thermocline depth, central wind stress and eastern Pacific SST yields the same picture and supports a recharge oscillator interpretation. The oscillation arises from the interaction between the recharge time of the Warm Pool and the time delay between east and west Pacific. Both finite Kelvin wave speed and SST dynamics contribute to the time delay. Including seasonality in the description, we find two periods of relative instability: boreal spring, with a large phase progression, and autumn, with nearly stationary phase.
Modeling of airglow and ionospheric parameters at Arecibo during quiet and disturbed periods in October 2002
Authors: Vlasov Michael N. , Nicolls Michael J. , Kelley Michael C. , Smith Steven M. , Aponte Néstor , González Sixto A.
Published: July 2005

The main purpose of this paper is to analyze observations of the 630.0‐nm (red line) and 557.7‐nm (green line) zenith airglow intensities measured during the month of October 2002 over Arecibo, Puerto Rico. We begin by describing an improved model for calculating the intensity of the red and green airglow lines that takes into account the role of molecular ions. We show, however, that at least for the data used in this study, it is not necessary to include the effects of molecular ions in our calculations. From observations of the airglow emissions on quiet days, we infer the general characteristics of the red‐line intensities, which show a minimum before midnight and a peak after midnight. These results are consistent with a decrease in NmF2 and an increase in hmF2 before midnight, followed by the so‐called “midnight collapse.” We then focus on the storm day of October 1–2, 2002, during which large‐amplitude variations in both the red‐ and green‐line intensities were observed and reproduced by the airglow model. An additional peak of NmF2, together with an hmF2 decrease, was observed before midnight, associated with the passage of a large‐scale atmospheric gravity wave. The NmF2 nighttime increase requires plasma flux from the plasmasphere, which we find can be as large as 109 cm−2 s−1. The plasmasphere needs an additional source of plasma in order to provide such a large flux, and we explain this by considering the role of the meridional wind in the plasma exchange process. Strong changes in the shape of the F2 region were observed during the downward and upward motion of the F2 layer during the storm period. We have found simple analytical solutions for the height distributions of the electron density in the F2 region by including the effects of recombination and diffusion. These height distributions are in excellent agreement with the measured profiles. Finally, we discuss mesospheric green‐line fluctuations and show that good agreement can be obtained for the storm conditions if small adjustments are made to the eddy diffusion coefficients in the mesosphere, which we associate with the passage of the gravity wave. However, we find that some green‐line behavior during quiet‐time conditions is difficult to explain.
Effects of urban development in the Puget Lowland, Washington, on interannual streamflow patterns: Consequences for channel form and streambed disturbance
Authors: Konrad Christopher P. , Booth Derek B. , Burges Stephen J.
Published: July 2005

Recovery and protection of streams in urban areas depend on a comprehensive understanding of how human activities affect stream ecosystems. The hydrologic effects of urban development and the consequences for stream channel form and streambed stability were examined in 16 streams in the Puget Lowland, Washington, using three streamflow metrics that integrate storm‐scale effects of urban development over annual to decadal timescales: the fraction of time that streamflow exceeds the mean streamflow (TQmean), the coefficient of variation of annual maximum streamflow (CVAMF), and the fraction of time that streamflow exceeds the 0.5‐year flood (T0.5). Urban streams had low interannual variability in annual maximum streamflow and brief duration of frequent high flows, as indicated by significant correlations between road density and both CVAMF and T0.5. The broader distribution of streamflow indicated by TQmean may be affected by urban development, but differences in TQmean between streams are also likely a result of other physiographic factors. The increase in the magnitude of frequent high flows due to urban development but not their cumulative duration has important consequences for channel form and bed stability in gravel bed streams because geomorphic equilibrium depends on moderate duration streamflow (e.g., exceeded 10% of the time). Streams with low values of TQmean and T0.5 are narrower than expected from hydraulic geometry. Dimensionless boundary shear stress (t*) for the 0.5‐year flood was inversely related to T0.5 among the streams, indicating frequent and extensive bed disturbance in streams with low values of T0.5. Although stream channels expand and the size of bed material increases in response to urban streamflow patterns, these adjustments may be insufficient to reestablish the disturbance regime in urban streams because of the differential increase in the magnitude of frequent high flows causing disturbance relative to any changes in longer duration, moderate flows that establish a stable channel.
Regulation of the poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and Sp3 is under the influence of cell density in primary cultured cells
Authors: Zaniolo Karine , Rufiange Anne , Leclerc Steeve , Desnoyers Serge , Guérin Sylvain L.
Published: July 2005

PARP-1 [poly(ADP-ribose) polymerase-1) is a nuclear enzyme that is involved in several cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity directed by the PARP-1 gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3 (where Sp is specificity protein). In the present study, we investigated whether (i) both PARP-1 expression and PARP-1 enzymatic activity are under the influence of cell density in primary cultured cells, and (ii) whether its pattern of expression is co-ordinated with that of Sp1/Sp3 at varying cell densities and upon cell passages. All types of cultured cells expressed PARP-1 in Western blot when grown to sub-confluence. However, a dramatic reduction was observed at post-confluence. Similarly, high levels of Sp1/Sp3 were observed by both Western blot and EMSAs (electrophoretic mobility-shift assays) in sub-confluent, but not post-confluent, cells. Consistent with these results, the promoter of the rPARP-1 (rat PARP-1) gene directed high levels of activity in sub-confluent, but not confluent, cells upon transfection of various CAT (chloramphenicol acetyltransferase)–rPARP-1 promoter constructs into cultured cells. The positive regulatory influence of Sp1 was not solely exerted on the rPARP-1 promoter constructs, as inhibition of endogenous Sp1 expression in HDKs (human dermal keratinocytes) through the transfection of Sp1 RNAi (RNA interference) considerably reduced endogenous hPARP-1 (human PARP-1) expression as well. The reduction in PARP-1 protein expression as cells reached confluence also translated into a corresponding reduction in PARP-1 activity. In addition, expression of both Sp1/Sp3, as well as that of PARP-1, was dramatically reduced as cells were passaged in culture and progressed towards irreversible terminal differentiation. PARP-1 gene expression therefore appears to be co-ordinated with that of Sp1 and Sp3 in primary cultured cells, suggesting that PARP-1 may play some important functions during the proliferative burst that characterizes wound healing.
Structure–activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II
Authors: Kelly Julie A. , Scalabrino Gaia A. , Slator Gillian R. , Cullen Aoife A. , Gilmer John F. , Lloyd David G. , Bennett Geoffrey W. , Bauer Karl , Tipton Keith F. , Williams Carvell H.
Published: July 2005

Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 μM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure–activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2′ subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics.
Observation of a tetrahedral reaction intermediate in the HIV-1 protease–substrate complex
Authors: Kumar Mukesh , Prashar Vishal , Mahale Smita , Hosur Madhusoodan V.
Published: July 2005

HIV-1 protease is an effective target for the design of drugs against AIDS. To help this process of drug design, three-dimensional structures have been determined of complexes between HIV-1 protease and a variety of transition-state analogue inhibitors. The true transition state, however, has not been structurally characterized. The crystal structure of the C95M/C1095A HIV-1 protease tethered dimer shows a distinctive feature in which the two flaps of the enzyme are in a ‘closed conformation’ even in the unliganded state. This unique feature has been utilized here to study the structure of HIV-1 protease complexed to an oligopeptide substrate of amino acid sequence His-Lys-Ala-Arg-Val-Leu*NPhe-Glu-Ala-Nle-Ser (where* denotes the cleavage site, and NPhe and Nle denote p-nitrophenylalanine and norleucine residues respectively). The X-ray structure of the complex refined against 2.03 Å (0.203 nm) resolution synchrotron data shows that the substrate is trapped as a tetrahedral reaction intermediate in the crystal. The hydrogen-bonding interactions between the reaction intermediate and the catalytic aspartates are different from those observed previously using transition-state analogues. The reaction intermediate did not dissociate to release the products, possibly due to the inflexibility introduced in the flaps when the enzyme is packed inside crystals.
Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling
Authors: Wu Zhengliang L. , Lech Miroslaw
Published: July 2005

Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules.
The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus
Authors: Allen James W. A. , Leach Nicholas , Ferguson Stuart J.
Published: July 2005

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.
Protein tyrosine phosphatase hPTPN20a is targeted to sites of actin polymerization
Authors: Fodero-Tavoletti Michelle T. , Hardy Matthew P. , Cornell Brent , Katsis Frosa , Sadek Christine M. , Mitchell Christina A. , Kemp Bruce E. , Tiganis Tony
Published: July 2005

The human genome encodes 38 classical tyrosine-specific PTPs (protein tyrosine phosphatases). Many PTPs have been shown to regulate fundamental cellular processes and several are mutated in human diseases. We report that the product of the PTPN20 gene at the chromosome locus 10q11.2 is alternatively spliced to generate 16 possible variants of the classical human non-transmembrane PTP 20 (hPTPN20). One of these variants, hPTPN20a, was expressed in a wide range of both normal and transformed cell lines. The catalytic domain of hPTPN20 exhibited catalytic activity towards tyrosyl phosphorylated substrates, confirming that it is a bona fide PTP. In serum-starved COS1 cells, hPTPN20a was targeted to the nucleus and the microtubule network, colocalizing with the microtubule-organizing centre and intracellular membrane compartments, including the endoplasmic reticulum and the Golgi apparatus. Stimulation of cells with epidermal growth factor, osmotic shock, pervanadate, or integrin ligation targeted hPTPN20a to actin-rich structures that included membrane ruffles. The present study identifies hPTPN20a as a novel and widely expressed phosphatase with a dynamic subcellular distribution that is targeted to sites of actin polymerization.
Evidence that the cADPR signalling pathway controls calcium-mediated microneme secretion in Toxoplasma gondii
Authors: Chini Eduardo N. , Nagamune Kisaburo , Wetzel Dawn M. , Sibley L. David
Published: July 2005

The protozoan parasite Toxoplasma gondii relies on calcium-mediated exocytosis to secrete adhesins on to its surface where they can engage host cell receptors. Increases in intracellular calcium occur in response to Ins(1,4,5)P3 and caffeine, an agonist of ryanodine-responsive calcium-release channels. We examined lysates and microsomes of T. gondii and detected evidence of cADPR (cyclic ADP ribose) cyclase and hydrolase activities, the two enzymes that control the second messenger cADPR, which causes calcium release from RyR (ryanodine receptor). We also detected endogenous levels of cADPR in extracts of T. gondii. Furthermore, T. gondii microsomes that were loaded with 45Ca2+ released calcium when treated with cADPR, and the RyR antagonists 8-bromo-cADPR and Ruthenium Red blocked this response. Although T. gondii microsomes also responded to Ins(1,4,5)P3, the inhibition profiles of these calcium-release channels were mutually exclusive. The RyR antagonists 8-bromo-cADPR and dantrolene inhibited protein secretion and motility in live parasites. These results indicate that RyR calcium-release channels that respond to the second-messenger cADPR play an important role in regulating intracellular Ca2+, and hence host cell invasion, in protozoan parasites.
Physical and functional interactions between members of the tumour suppressor p53 and the Sp families of transcription factors: importance for the regulation of genes involved in cell-cycle arrest and apoptosis
Authors: Koutsodontis George , Vasilaki Eleftheria , Chou Wan-Chih , Papakosta Paraskevi , Kardassis Dimitris
Published: July 2005

In the present study, we have investigated mechanisms of transcriptional co-operation between proteins that belong to the tumour suppressor p53 and Sp (specificity protein) families of transcription factors. Such mechanisms may play an important role in the regulation of genes containing binding sites for both classes of transcription factors in their promoters. Two of these genes were analysed in the present study: the cyclin-dependent kinase inhibitor p21Cip1 gene and the PUMA (p53-up-regulated mediator of apoptosis) gene. We found that Sp1 and Sp3, but not Sp2, co-operate functionally with p53, p73 and p63 for the synergistic transactivation of the p21Cip1 promoter in Drosophila Schneider SL2 cells that lack endogenous Sp factors. We also found that Sp1 strongly transactivated the PUMA promoter synergistically with p53, whereas deletion of the Sp1-binding sites abolished the transactivation by p53. Using p53 mutant forms in GST (glutathione S-transferase) pull-down assays, we found that the C-terminal 101 amino acids of p53, which include the oligomerization and regulatory domains of the protein, are required for the physical interactions with Sp1 and Sp3, and that deletion of this region abolished transactivation of the p21Cip1 promoter. Utilizing truncated forms of Sp1, we established that p53 interacted with the two transactivation domains A and B, as well as the DNA-binding domain. Our findings suggest that Sp factors are essential for the cellular responses to p53 activation by genotoxic stress. Understanding in detail how members of the p53 and Sp families of transcription factors interact and work together in the p53-mediated cellular responses may open new horizons in cancer chemotherapy.
E3 ubiquitin ligase RNF2 interacts with the S6′ proteasomal ATPase subunit and increases the ATP hydrolysis activity of S6′
Authors: Lee Sun-Joo , Choi Dongwon , Rhim Hyangshuk , Kang Seongman
Published: July 2005

We reported previously that the human RNF2 (RING finger protein 2) protein is an E3 ubiquitin ligase that interacts with the human ubiquitin-conjugating enzyme Hip-2/hE2-25K. In the present study, we show that RNF2 interacts with S6′ ATPase, a subunit of the proteasomal 19 S regulatory complex. S6′ interacts with RNF2 through its N-terminal RING domain, and RNF2 interacts with S6′ through its C-terminal region. Interestingly, the RNF2-S6′ interaction increases the ATP hydrolysis activity of the S6′ protein. Moreover, S6′ ATPase activity is highly increased in the presence of ubiquitinated proteins. The present study suggests that the E3 ubiquitin ligase RNF2 might have a dual function: facilitating the ubiquitination of its target substrates and recruiting the substrates to the proteasome. Furthermore, ATP hydrolysis in the E3/proteasome complex might act as an important signal for the protein degradation pathway.
Nep1, a Schizosaccharomyces pombe deneddylating enzyme
Authors: Zhou Lihong , Watts Felicity Z.
Published: July 2005

Nedd8 is a ubiquitin-like modifier that is attached to the cullin components of E3 ubiquitin ligases. More recently, p53 has also been shown to be Nedd8-modified. Nedd8 attachment occurs in a manner similar to that observed for other ubiquitin-like modifiers. In the present study, we report on the characterization of Nep1, a deneddylating enzyme in fission yeast (Schizosaccharomyces pombe). Unlike loss of ned8, deletion of the nep1 gene is not lethal, although nep1.d cells are heterogeneous in length, suggesting a defect in cell-cycle progression. Viability of nep1.d cells is dependent on a functional spindle checkpoint but not on the DNA integrity checkpoint. Deletion of a related gene (nep2), either alone or in combination with nep1.d, also has little effect on cell viability. We show that Nep1 can deneddylate the Pcu1, Pcu3 and Pcu4 cullins in vitro and that its activity is sensitive to N-ethylmaleimide, consistent with the idea that it is a member of the cysteine protease family. nep1.d cells accumulate Nedd8-modified proteins, although these do not correspond to modified forms of the cullins, suggesting that, although Nep1 can deneddylate cullins in vitro, this is not its main function in vivo. Nep1 can be co-precipitated with the signalosome subunit Csn5. Nep1 itself is present in a high-molecular-mass complex, but the presence of this complex is not dependent on the production of intact signalosomes. Our results suggest that, in vivo, Nep1 may be responsible for deneddylating proteins other than cullins.
Interleukin-1β induces death in chondrocyte-like ATDC5 cells through mitochondrial dysfunction and energy depletion in a reactive nitrogen and oxygen species-dependent manner
Authors: Yasuhara Rika , Miyamoto Yoichi , Akaike Takaaki , Akuta Teruo , Nakamura Masanori , Takami Masamichi , Morimura Naoko , Yasu Kayoko , Kamijo Ryutaro
Published: July 2005

IL-1 (interleukin-1) acts as a key mediator of the degeneration of articular cartilage in RA (rheumatoid arthritis) and OA (osteoarthritis), where chondrocyte death is observed. It is still controversial, however, whether IL-1 induces chondrocyte death. In the present study, the viability of mouse chondrocyte-like ATDC5 cells was reduced by the treatment with IL-1β for 48 h or longer. IL-1β augmented the expression of the catalytic gp91 subunit of NADPH oxidase, gp91phox, as well as inducible NO synthase in ATDC5 cells. Generation of nitrated guanosine and tyrosine suggested the formation of reactive nitrogen species including ONOO− (peroxynitrite), a reaction product of NO and O2−, in ATDC5 cells and rat primary chondrocytes treated with IL-1β. Death of ATDC5 cells after IL-1β treatment was prevented by an NADPH-oxidase inhibitor, AEBSF [4-(2-aminoethyl)benzenesulphonyl fluoride], an NO synthase inhibitor, L-NAME (NG-nitro-L-arginine methyl ester), and a ONOO− scavenger, uric acid. The viability of ATDC5 cells was reduced by the ONOO−-generator 3-(4-morpholinyl)sydnonimine hydrochloride, but not by either the NO-donor 1-hydroxy-2-oxo-3-(N-methyl-2-aminopropyl)-3-methyl-1-triazene or S-nitrosoglutathione. Disruption of mitochondrial membrane potential and ATP deprivation were observed in IL-1β-treated ATDC5 cells, both of which were restored by L-NAME, AEBSF or uric acid. On the other hand, no morphological or biochemical signs indicating apoptosis were observed in these cells. These results suggest that the death of chondrocyte-like ATDC5 cells was mediated at least in part by mitochondrial dysfunction and energy depletion through ONOO− formation after IL-1β treatment.
Deletions within a CA-repeat-rich region of intron 4 of the human SP-B gene affect mRNA splicing
Authors: Lin Zhenwu , Thomas Neal J. , Wang Yunhua , Guo Xiaoxuan , Seifart Carola , Shakoor Hasan , Floros Joanna
Published: July 2005

Length variants within a CA-repeat-rich region of intron 4 of the human SP-B (pulmonary surfactant protein-B) gene are associated with several lung diseases. The hypothesis that SP-B intron 4 affects mRNA splicing was studied. SP-B minigenes containing exons 1–6 with a normal-sized intron 4 (pBi4normal) or intron 4 containing deletions (pBi4del) of 193, 211, 264 or 340 bp were expressed in CHO (Chinese hamster ovary) cells by transient transfection. Two forms of SP-B transcripts, normal and incompletely spliced, were detected. With pBi4normal, normal-sized SP-B mRNA was the predominant form and a very low amount of incompletely spliced mRNA was present, whereas with the pBi4del variants the amount of normal SP-B mRNAs was lower and the amount of incompletely spliced mRNA was relatively high. Reverse transcription–PCR results and sequencing data indicated that the incompletely spliced SP-B RNA contained intron 4 sequence, and this incompletely spliced RNA was also observed in normal lung. Lung cancer tissues with intron 4 deletions exhibited a larger amount of abnormally spliced RNAs compared with normal lung tissue or cancerous tissue with normal-sized intron 4. The results indicate that intron 4 length variants affect SP-B mRNA splicing, and that this may contribute to lung disease.
Thiol–disulphide interchange in tubulin: kinetics and the effect on polymerization
Authors: Britto P. J. , Knipling Leslie , Mcphie Peter , Wolff J.
Published: July 2005

All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] and 2,2′-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulphide products react further to form both intra- and intermonomer disulphide bonds that can be reversed by reducing agents. With MMTS (methyl methanethiosulphonate) or ODNB (n-octyl-dithio-2-nitrobenzoate), virtually no protein–protein disulphide bonds are formed and the ODNB reaction can be given as the sum of three exponential terms with pseudo-first-order rate constants of 0.206, 0.069 and 0.010 s−1 at pH 6.5, suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of the helix content. MMTS-induced loss of SH groups leads to progressive increases in the critical concentration and loss of polymerization competence that can be reversed by assembly promoters such as higher protein concentration, taxol or high ionic strength. Under such conditions, the substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles.
Up-regulation of phospholipid hydroperoxide glutathione peroxidase in rat casein-induced polymorphonuclear neutrophils
Authors: Hattori Hiroyuki , Imai Hirotaka , Hanamoto Akiharu , Furuhama Kazuhisa , Nakagawa Yasuhito
Published: July 2005

Antioxidant enzymes play key roles in the protection of cells from oxidative damage. Little is known, however, about the expression of antioxidants and/or their roles in PMNs (polymorphonuclear leucocytes), which are thought to suffer from oxidative stress in an inflammation site. In the present paper, we report on the regulation of expression of PHGPx (phospholipid hydroperoxide glutathione peroxidase) and cGPx (cytosolic glutathione peroxidase) in rat PMNs in the inflammation site. PHGPx mRNA levels were much lower in casein-induced peritoneal and carrageenan-induced pleural PMNs just after their collection than in peripheral PMNs. cGPx mRNA was also reduced in the casein-induced PMNs, but not in carrageenan-induced PMNs. Both enzymes with decreased levels in the casein-induced PMNs were up-regulated during further 24 h cultivation in vitro and in vivo, with elevation of their protein levels and activities, and reduction of intracellular peroxides. Up-regulation of PHGPx mRNA was attenuated by cycloheximide, a protein synthesis inhibitor, and this effect was cancelled by culturing the cells in the conditioned medium of the cultured casein-induced PMNs. This latter effect was attenuated by pre-treatment with anti-GRO (growth-regulated oncogene) antibody. Recombinant rat GRO could also induce the up-regulation in the presence of cycloheximide, demonstrating that GRO may play an important role in the PHGPx up-regulation of casein-induced PMNs. Production of the lipid mediators leukotriene B4 and 5-HETE (5-hydroxyeicosatetraenoic acid) was decreased in the cultured casein-induced PMNs exhibiting PHGPx up-regulation. The evidence obtained indicates that PHGPx activity in the activated PMNs would be related to the appearance of the intrinsic function of PMNs in the inflammatory site.
Down-regulation of Rap1 activity is involved in ephrinB1-induced cell contraction
Authors: Riedl Jurgen A. , Brandt Dominique T. , Batlle Eduard , Price Leo S. , Clevers Hans , Bos Johannes L.
Published: July 2005

Ephrins are cell surface ligands that activate Eph receptor tyrosine kinases. This ligand–receptor interaction plays a central role in the sorting of cells. We have previously shown that the ephrinB–EphB signalling pathway is also involved in the migration of intestinal precursor cells along the crypts. Using the colon cell line DLD1 expressing the EphB2 receptor, we showed that stimulation of these cells with soluble ephrinB1 results in a rapid retraction of cell extensions and a detachment of cells. On ephrinB1 stimulation, the small GTPases Rho and Ras are activated and Rap1 is inactivated. Importantly, when a constitutively active Rap1 mutant was introduced into these cells, ephrinB1-induced retraction was inhibited. From these results, we conclude that down-regulation of Rap1 is a prerequisite for ephrin-induced cell retraction in colon cells.
Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo
Authors: Soni Rajesh K. , Mehra Parul , Mukhopadhyay Gauranga , Kumar Dhar Suman
Published: July 2005

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.