bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermosphere density variations due to the 15–24 April 2002 solar events from CHAMP/STAR accelerometer measurements
Authors: Forbes Jeffrey M. , Lu Gang , Bruinsma Sean , Nerem Steven , Zhang Xiaoli
Published: December 2005

Thermosphere densities near 410 km altitude between ±87° latitude and near 0430 and 1530 local time from the accelerometer experiment on the CHAMP satellite are used to elucidate the response to three coronal mass ejections occurring on 17, 19, and 21 April 2002. Comparisons of the global responses with the NRLMSISE00 empirical model and the NCAR TIEGCM are performed and interpreted. An enhanced daytime response in comparison to TIEGCM on 17 April is found that is suggestive of preconditioning of the atmosphere due to enhanced solar EUV fluxes maximizing on 13 April. Out of several solar wind parameters and geophysical indices that were examined, the highest correlation with thermosphere densities occurred with respect to the magnitude of the interplanetary magnetic field as measured by the ACE spacecraft. Wave‐like structures with scales ranging from 100s to 1000s of kilometers are also revealed in the CHAMP data. These structures are primarily a nighttime phenomena, and the associated total variance is correlated with magnetic activity throughout the 15–24 April 2002 period. The NCAR TIEGCM was utilized to provide the basis for interpreting the equatorward propagation of large‐scale traveling atmospheric disturbance (TADs). Following a sudden increase in magnetic activity and high‐latitude heating, TADs were launched from both hemispheres, traveled toward the equator with phase speeds of order 800 m s−1, constructively interfered near the equator to produce a total density perturbation of ∼20%, and then passed through each other and into the opposite hemisphere. Perspectives on future applications of CHAMP accelerometer data to elucidate magnetic storm‐related perturbations of the thermosphere are outlined.
Search for past life on Mars: Physical and chemical characterization of minerals of biotic and abiotic origin: part 1 ‐ Calcite
Authors: Stalport Fabien , Coll Patrice , Cabane Michel , Person Alain , González Rafael Navarro , Raulin Francois , Vaulay Marie Jo , Ausset Patrick , McKay Chris P. , Szopa Cyril , Zarnecki John
Published: December 2005

Several lines of evidence suggest that early Mars once had liquid water on its surface, a denser atmosphere and a mild climate. Similar environmental conditions led to the origin of life on the Earth more than 3.5 billion years ago; consequently, life might also have originated on Mars. The Viking landers searched for evidence of organic molecules on the surface of Mars, and found that the Martian soil is depleted in organics at ppb levels at the landing sites. We contend that inorganic compounds could give us interesting clues as to the existence of possible biological activity in future astrobiological missions to Mars. Consequently, we have investigated the physical and chemical properties of calcite, which could be expected on Mars because liquid water was certainly present on the surface of early Mars and carbon dioxide was abundant in its atmosphere. Calcite is interesting because on Earth this mineral is produced by abiotic processes as well as by biological activity. One may suppose that crystalline defects and trace element in the crystal lattice and the growth speed of biotic calcites must indicate a difference between them and pure abiotic calcites. We investigated twelve different terrestrial calcite samples from various origins: biotic, diagenetic and abiotic. The minerals were studied by X‐ray diffraction and electron scanning microscopy to determine their mineralogical and chemical composition, and differential thermal analysis coupled to thermogravimetric analysis (DTA‐TG) to determine their thermal behavior. Our results show that the thermal degradation of abiotic calcite starts at a temperature at least 40°C higher than the degradation temperature of any biotic calcite investigated. Consequently, in the case of a Martian in‐situ study or in a sample return mission, the analysis of Martian minerals by DTA‐TG represents a promising approach to detect evidence of past biological activity on Mars.
Estimating 2 × CO2 warming in an aquaplanet GCM using the fluctuation‐dissipation theorem
Authors: Langen Peter L. , Alexeev Vladimir A.
Published: December 2005

A statistical analysis based on the fluctuation‐dissipation theorem (FDT) is applied to estimate a model response to a small external forcing. An atmospheric GCM (the NCAR CCM3) coupled to an oceanic upper mixed layer is used to produce a long equilibrium run with 1 × CO2 concentration. Exploiting the FDT, we use the statistical properties of the internal variability of the system to approximately restore the linearized operator of the system. The modes of this operator are analyzed and compared with those calculated using the approach based on a perturbation method employed earlier by one of the authors. The sensitivity of the model to 2 × CO2 forcing calculated using the FDT approach compares well with the results obtained in an actual 2 × CO2 run.
Equatorial electron density measurements in Saturn's inner magnetosphere
Authors: Persoon A. M. , Gurnett D. A. , Kurth W. S. , Hospodarsky G. B. , Groene J. B. , Canu P. , Dougherty M. K.
Published: December 2005

Upper hybrid resonance emissions detected by the Radio and Plasma Wave Science (RPWS) instrument on the Cassini spacecraft are used to obtain electron densities on five equatorial orbits of Saturn at radial distances ranging from 3 to 9 saturnian radii (RS). The electron density profiles for these orbits show a highly repeatable radial dependence beyond 5 RS, decreasing with increasing radial distance approximately as (1/R)3.63. Inside 5 RS, the electron density profiles are highly variable. We show that these radial variations are consistent with a centrifugally‐driven outward transport of plasma from a source inside 5 RS.
Probing upper thermospheric neutral densities at Mars using electron reflectometry
Authors: Lillis R. J. , Engel J. H. , Mitchell D. L. , Brain D. A. , Lin R. P. , Bougher S.W. , Acuña M. H.
Published: December 2005

We present a new technique for inferring neutral densities in the Martian upper atmosphere from atmospheric absorption of magnetically reflected solar wind electrons. Using electron loss cone measurements from the Magnetometer/Electron Reflectometer (MAG/ER) experiment on board Mars Global Surveyor (MGS), we derive upper thermospheric (∼160–230 km altitude) densities in the southern hemisphere from 160° to 200°E at 2am local time, continuously from 1999 to 2005. We find a mean density of 0.027 kg/km3 at 160 km and observe a latitude‐dependent, repeatable seasonal variation of a factor of 1.8–4, with inter‐annual differences and consistently lower winter densities at 50°–55°S, compared with 0°–30°S. The mean densities, overall seasonal variation and latitude dependence are in general agreement with the Mars Thermosphere Global Circulation Model (MTGCM). These measurements are important for improved understanding of the dynamics of Mars' upper atmosphere and for planning spacecraft aerobraking maneuvers.
Detecting submarine groundwater discharge with synoptic surveys of sediment resistivity, radium, and salinity
Authors: Breier J. A. , Breier C. F. , Edmonds H. N.
Published: December 2005

A synoptic geophysical and geochemical survey was used to investigate the occurrence and spatial distribution of submarine discharges of water to upper Nueces Bay, Texas. The 17 km survey incorporated continuous resistivity profiling; measurements of surface water salinity, temperature, and dissolved oxygen; and point measurements of dissolved Ra isotopes. The survey revealed areas of interleaving, vertical fingers of high and low conductivity extending up through 7 m of bay bottom sediments into the surface water, located within 100 m of surface salinity and dissolved Ra maxima along with peaks in water temperature and lows in dissolved oxygen. These results indicate either brackish submarine groundwater discharge or the leakage of oil field brine from submerged petroleum pipelines.
Microwave signatures over carbonate sedimentary platforms in arid areas: Potential geological applications of passive microwave observations?
Authors: Prigent Catherine , Munier Jean‐Marie , Thomas Bertrand , Ruffié Gilles
Published: December 2005

Microwave emissivities have been calculated over the globe from satellite observations between 19 and 85 GHz with the Special Sensor Microwave/Imager (SSM/I). Unusually low and stable microwave emissivities are observed in Oman and Egypt over large regions. Spatial patterns of low emissivities are well correlated with the distribution of tertiary sediments. Dielectric measurements have been performed and confirm that carbonate rocks have high permittivities (higher than 8 as compared to typical values of 5 for other rocks or 2.5 for silicate sands) and this explains the observed low emissivities.
Precession of the whistler polarisation plane normal observed on Freja
Authors: Karlsson R. L. , Carozzi T. D. , Bergman J. E. S. , Eriksson A. I.
Published: December 2005

We have observed that the normal to the polarisation plane of a whistler precesses around the geomagnetic field. Magnetic field observations by the Freja spacecraft have been used. These results were made possible by the use of an analysis which estimates the instantaneous polarisation properties of waves. During one event, the instantaneous normal of the polarisation plane precesses around the direction antiparallel to the geomagnetic field with an average deviation of 6°. In total, the normal precesses four times around this direction as the whistler frequency decreases from 2 to 0.4 kHz in about 0.2 s. Available theoretical understanding can not unambiguously explain the precession. We show that the precession of whistler wave vectors can be observed, and suggest to use this new observable parameter to put new constraints on the theories of whistler propagation.
Energy transport in the thermosphere during the solar storms of April 2002
Authors: Mlynczak Martin G. , Martin‐Torres F. Javier , Crowley Geoff , Kratz David P. , Funke Bernd , Lu Gang , Lopez‐Puertas Manuel , Russell James M. , Kozyra Janet , Mertens Chris , Sharma Ramesh , Gordley Larry , Picard Richard , Winick Jeremy , Paxton Larry
Published: December 2005

The dramatic solar storm events of April 2002 deposited a large amount of energy into the Earth's upper atmosphere, substantially altering the thermal structure, the chemical composition, the dynamics, and the radiative environment. We examine the flow of energy within the thermosphere during this storm period from the perspective of infrared radiation transport and heat conduction. Observations from the SABER instrument on the TIMED satellite are coupled with computations based on the ASPEN thermospheric general circulation model to assess the energy flow. The dominant radiative response is associated with dramatically enhanced infrared emission from nitric oxide at 5.3 μm from which a total of ∼7.7 × 1023 ergs of energy are radiated during the storm. Energy loss rates due to NO emission exceed 2200 Kelvin per day. In contrast, energy loss from carbon dioxide emission at 15 μm is only ∼2.3% that of nitric oxide. Atomic oxygen emission at 63 μm is essentially constant during the storm. Energy loss from molecular heat conduction may be as large as 3.8% of the NO emission. These results confirm the “natural thermostat” effect of nitric oxide emission as the primary mechanism by which storm energy is lost from the thermosphere below 210 km.
A performance evaluation of the operational Jet Propulsion Laboratory/University of Southern California Global Assimilation Ionospheric Model (JPL/USC GAIM)
Authors: Mandrake L. , Wilson B. , Wang C. , Hajj G. , Mannucci A. , Pi X.
Published: December 2005

The Jet Propulsion Laboratory/University of Southern California Global Assimilation Ionospheric Model (JPL/USC GAIM) uses two data assimilation techniques to optimally combine ionospheric measurements with the physics model: a sparse, traditional Kalman filter to estimate the three‐dimensional density state, and a four‐dimensional variational approach (4DVAR) to estimate ionospheric drivers such as the equatorial E × B drift or neutral winds. In this paper we study a specific implementation of the JPL/USC GAIM Kalman filter (single ion, low‐resolution, and input data from 200 ground GPS sites) and validate its global accuracy over 137 days by comparisons to independent GPS slant total electron content (TEC) observations (“missing site” tests) and independent JASON vertical TEC observations. The assimilation accuracy is robust with a slant TEC spatial prediction RMS error of 4 TECU (Total Electron Content Unit, 1 × 1016 e‐/m2) on average and a vertical TEC JASON RMS error of 7 TECU. Removing what appears to be a positive ≈4.4 TECU bias from the JASON observations, we obtain an improved performance of 5.3 TECU over the oceans. Comparisons with a single, thin shell global ionospheric map model and the International Reference Ionosphere and Bent ionospheric models are also provided.
Basic properties of anisotropic stellar wind expansion in the fluid approach
Authors: Hubert D. , Leblanc F.
Published: December 2005

Whereas in an isotropic temperature atmosphere both the hydrostatic equation and the momentum equation give the same conditions for hydrostatic equilibrium, in the anisotropic case the situation is ambiguous. It is found that for an anisotropic temperature the hydrostatic equilibrium conditions have to be deduced from the momentum equation. The condition for hydrostatic equilibrium is that both the parallel and the perpendicular temperatures, to the radial direction, decrease more rapidly than 1/r in the general case and that the perpendicular temperature decreases more rapidly than 1/r when the parallel temperature is constant. The momentum equation displays a transonic solution when at least one of the temperatures, parallel or perpendicular to the radial direction, decreases less rapidly than 1/r in the general case and when the perpendicular temperature decreases less rapidly than 1/r when the parallel temperature is constant. In an anisotropic atmosphere the parallel thermal velocity is the critical velocity. The properties of the transonic expansion in an isothermal and anisotropic atmosphere are studied. The initial velocity, the critical distance position, the terminal velocity, and the density profile are significantly different from the isotropic case. These properties are opposite with respect to the value of the anisotropy T⊥/T∥ > 1.0 and T⊥/T∥ < 1.0. In particular, for a perpendicular temperature larger than the parallel temperature the acceleration starts really at a significant distance from the base of the atmosphere, whereas the critical point is closer to the base of the atmosphere compared to the isotropic case. For an opposite anisotropy the transonic point moves far away from the base of the atmosphere, and the terminal velocity is significantly decreased for small temperature anisotropy. The mass loss rate is drastically affected for an anisotropy T⊥/T∥ > 2.0. The extension of this study to multimoment anisotropic models can be useful for the interpretation of particle simulation of rarefied stellar atmosphere expansion.
An event‐based approach to validating solar wind speed predictions: High‐speed enhancements in the Wang‐Sheeley‐Arge model
Authors: Owens M. J. , Arge C. N. , Spence H. E. , Pembroke A.
Published: December 2005

One of the primary goals of the Center for Integrated Space Weather Modeling (CISM) effort is to assess and improve prediction of the solar wind conditions in near‐Earth space, arising from both quasi‐steady and transient structures. We compare 8 years of L1 in situ observations to predictions of the solar wind speed made by the Wang‐Sheeley‐Arge (WSA) empirical model. The mean‐square error (MSE) between the observed and model predictions is used to reach a number of useful conclusions: there is no systematic lag in the WSA predictions, the MSE is found to be highest at solar minimum and lowest during the rise to solar maximum, and the optimal lead time for 1 AU solar wind speed predictions is found to be 3 days. However, MSE is shown to frequently be an inadequate “figure of merit” for assessing solar wind speed predictions. A complementary, event‐based analysis technique is developed in which high‐speed enhancements (HSEs) are systematically selected and associated from observed and model time series. WSA model is validated using comparisons of the number of hit, missed, and false HSEs, along with the timing and speed magnitude errors between the forecasted and observed events. Morphological differences between the different HSE populations are investigated to aid interpretation of the results and improvements to the model. Finally, by defining discrete events in the time series, model predictions from above and below the ecliptic plane can be used to estimate an uncertainty in the predicted HSE arrival times.
Effects of the antifungal antibiotic clotrimazole on human cardiac repolarization potassium currents
Authors: Tian Miao , Dong Ming‐Qing , Chiu Shui‐Wha , Lau Chu‐Pak , Li Gui‐Rong
Published: December 2005

The antifungal antibiotic clotrimazole (CLT) shows therapeutic effects on cancer, sickle cell disease, malaria, etc. by inhibiting membrane intermediate‐conductance Ca2+‐activated K+channels (IKCa). However, it is unclear whether this drug would affect human cardiac K+currents. The present study was therefore designed to investigate the effects of CLT on transient outward K+current (Ito1), and ultra‐rapid delayed rectifier K+current (IKur) in isolated human atrial myocytes, and cloned hERG channel current (IhERG) and recombinant human cardiac KCNQ1/KCNE1 channel current (IKs) expressed in HEK 293 cells.

It was found that CLT inhibitedIto1with an IC50of 29.5 μM, acceleratedIto1inactivation, and decreased recovery ofIto1from inactivation. In addition, CLT inhibited human atrialIKurin a concentration‐dependent manner (IC50=7.6 μM).

CLT substantially suppressedIhERG(IC50=3.6 μM), and negatively shifted the activation conductance ofIhERG. Moreover, CLT inhibitedIKs(IC50=15.1 μM), and positively shifted the activation conductance of the current.

These results indicate that the antifungal antibiotic CLT substantially inhibits human cardiac repolarization K+currents includingIto1,IKur,IhERG, andIKs. However, caution is recommended when correlating the observedin vitroeffects on cardiac ion currents to the clinical relevance.

British Journal of Pharmacology(2006)147, 289–297. doi:10.1038/sj.bjp.0706590
Interactions of metoclopramide and ergotamine with human 5‐HT3A receptors and human 5‐HT reuptake carriers
Authors: Walkembach Jan , Brüss Michael , Urban Bernd W , Barann Martin
Published: December 2005

British Journal of Pharmacology (2005) 146, 1156. doi:10.1038/sj.bjp.0706610
PERK (eIF2α kinase) is required to activate the stress-activated MAPKs and induce the expression of immediate-early genes upon disruption of ER calcium homoeostasis
Authors: Liang Shun-Hsin , Zhang Wei , Mcgrath Barbara C. , Zhang Peichuan , Cavener Douglas R.
Published: December 2005

The eIF2α (eukaryotic initiation factor-2α) kinase PERK (doublestranded RNA-activated protein kinase-like ER kinase) is essential for the normal function of highly secretory cells in the pancreas and skeletal system, as well as the UPR (unfolded protein response) in mammalian cells. To delineate the regulatory machinery underlying PERK-dependent stress-responses, gene profiling was employed to assess global changes in gene expression in PERK-deficient MEFs (mouse embryonic fibroblasts). Several IE (immediate-early) genes, including c-myc, c-jun, egr-1 (early growth response factor-1), and fra-1 (fos-related antigen-1), displayed PERK-dependent expression in MEFs upon disruption of calcium homoeostasis by inhibiting the ER (endoplasmic reticulum) transmembrane SERCA (sarcoplasmic/ER Ca2+-ATPase) calcium pump. Induction of c-myc and egr-1 by other reagents that elicit the UPR, however, showed variable dependence upon PERK. Induction of c-myc expression by thapsigargin was shown to be linked to key signalling enzymes including PLC (phospholipase C), PI3K (phosphatidylinositol 3-kinase) and p38 MAPK (mitogen-activated protein kinase). Analysis of the phosphorylated status of major components in MAPK signalling pathways indicated that thapsigargin and DTT (dithiothreitol) but not tunicamycin could trigger the PERK-dependent activation of JNK (c-Jun N-terminal kinase) and p38 MAPK. However, activation of JNK and p38 MAPK by non-ER stress stimuli including UV irradiation, anisomycin, and TNF-α (tumour necrosis factor-α) was found to be independent of PERK. PERK plays a particularly important role in mediating the global cellular response to ER stress that is elicited by the depletion of calcium from the ER. We suggest that this specificity of PERK function in the UPR is an extension of the normal physiological function of PERK to act as a calcium sensor in the ER.
Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC
Authors: Matsumura Eitaro , Sakai Masashi , Hayashi Katsuaki , Murakami Shuichiro , Takenaka Shinji , Aoki Kenji
Published: December 2005

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.
Group V and X secretory phospholipase A2 prevents adenoviral infection in mammalian cells
Authors: Mitsuishi Michiko , Masuda Seiko , Kudo Ichiro , Murakami Makoto
Published: December 2005

sPLA2 (secretory phospholipase A2) enzymes have been implicated in various biological events, yet their precise physiological functions remain largely unresolved. In the present study we show that group V and X sPLA2s, which are two potent plasma membrane-acting sPLA2s, are capable of preventing host cells from being infected with an adenovirus. Bronchial epithelial cells and lung fibroblasts pre-expressing group V and X sPLA2s showed marked resistance to adenovirus-mediated gene delivery in a manner dependent on their catalytic activity. Although adenovirus particles were insensitive to recombinant group V and X sPLA2s, direct addition of these enzymes to 293A cells suppressed both number and size of adenovirus plaque formation. Group V and X sPLA2s retarded the entry of adenovirus into endosomes. Moreover, adenoviral infection was suppressed by LPC (lysophosphatidylcholine), a membrane-hydrolytic product of these sPLA2s. Thus hydrolysis of the plasma membrane by these sPLA2s may eventually lead to the protection of host cells from adenovirus entry. Given that group V and X sPLA2s are expressed in human airway epithelium and macrophages and that the expression of endogenous group V sPLA2 is upregulated by virus-related stimuli in these cells, our present results raise the possibility that group V and X sPLA2s may play a role in innate immunity against adenoviral infection in the respiratory tract.
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transcriptional regulation by Oct-1 in human endothelial cells: implications for atherosclerosis
Authors: Chen Jiawei , Liu Yong , Liu Hongmei , Hermonat Paul L. , Mehta Jawahar L.
Published: December 2005

LOX-1, a receptor for ox-LDL (oxidized low-density lipoprotein), has recently been determined to play a critical role in the progression of atherosclerosis. LOX-1 expression (mRNA and protein) has been shown to be up-regulated by pro-atherogenic stimuli, such as ox-LDL and Ang II (angiotensin II). However, the molecular mechanisms of these up-regulations are unclear. In the present study, we explored LOX-1 transcriptional promoter activation in response to ox-LDL and Ang II. Under basal states, LOX-1 core promoter (LOX-1 −35/+36) was found to be sufficient for its basal activity in HCAECs (human coronary artery endothelial cells). More importantly, we found that ox-LDL (60 μg/ml for 24 h) induced LOX-1 promoter activity significantly and that a 105 bp fragment (between nt −1599 and −1494) was required for this activation. Within this 106 bp fragment, there is a potential binding motif for the transcription factor Oct-1 (octamer-1). By electrophoretic mobility-shift assay, we observed the activation of Oct-1 by ox-LDL. The critical role of Oct-1 in ox-LDL-induced LOX-1 promoter activation was further confirmed by mutagenesis assay. For comparison, we also examined LOX-1 promoter activation in response to Ang II (1 μmol/l for 24 h). Interestingly, another promoter region, between nt −2336 and −1990, was required for Ang II-induced LOX-1 promoter activation. In conclusion, the present study strongly suggests that ox-LDL, by activating Oct-1, induces LOX-1 promoter activation. Furthermore, this study suggests that while ox-LDL and Ang II both induce LOX-1 expression in HCAECs, the underlying mechanisms of promoter activation are different from each other.
ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities
Authors: Osawa Hiroki , Stacey Gary , Gassmann Walter
Published: December 2005

A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5′-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.
Characterization of the gene encoding glyoxalase II from Leishmania donovani: a potential target for anti-parasite drugs
Authors: Padmanabhan Prasad K. , Mukherjee Angana , Madhubala Rentala
Published: December 2005

The glyoxalase system is a ubiquitous detoxification pathway that protects against cellular damage caused by highly reactive oxoaldehydes such as methylglyoxal which is mainly formed as a by-product of glycolysis. The gene encoding GLOII (glyoxalase II) has been cloned from Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. DNA sequence analysis revealed an ORF (open reading frame) of ∼888 bp that encodes a putative 295-amino-acid protein with a calculated molecular mass of 32.5 kDa and a predicted pI of 6.0. The sequence identity between human GLOII and LdGLOII (L. donovani GLOII) is only 35%. The ORF is a single-copy gene on a 0.6-Mb chromosome. A ∼38 kDa protein was obtained by heterologous expression of LdGLOII in Escherichia coli, and homogeneous enzyme was obtained after affinity purification. Recombinant L. donovani GLOII showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOII protein could detect a band of anticipated size ∼32 kDa in promastigote extracts. By overexpressing the GLOII gene in Leishmania donovani using Leishmania expression vector pspαhygroα, we detected elevated expression of GLOII RNA and protein. Overexpression of the GLOII gene will facilitate studies of gene function and its relevance as a chemotherapeutic target. This is the first report on the molecular characterization of glyoxalase II from Leishmania spp. The difference in the substrate specificity of the human and Leishmania donovani glyoxalase II enzyme could be exploited for structure-based drug design of selective inhibitors against the parasite.
Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene
Authors: De Luca Antonella , Sacchetta Paolo , Di Ilio Carmine , Favaloro Bartolo
Published: December 2005

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.
Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells
Authors: Omatsu-Kanbe Mariko , Inoue Kazuko , Fujii Yusuke , Yamamoto Takefumi , Isono Takahiro , Fujita Norihisa , Matsuura Hiroshi
Published: December 2005

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1–100 μM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.
The forkhead transcription factor Foxi1 directly activates the AE4 promoter
Authors: Kurth Ingo , Hentschke Moritz , Hentschke Suna , Borgmeyer Uwe , Gal Andreas , Hübner Christian A.
Published: December 2005

Intercalated cells are highly specialized cells within the renal collecting duct epithelium and play an important role in systemic acid–base homoeostasis. Whereas type A intercalated cells secrete protons via an apically localized H+-ATPase, type B intercalated cells secrete HCO3−. Type B intercalated cells specifically express the HCO3−/Cl− exchanger AE4 (anion exchanger 4), encoded by Slc4a9. Mice with a targeted disruption of the gene for the forkhead transcription factor Foxi1 display renal tubular acidosis due to an intercalated cell-differentiation defect. Collecting duct cells in these mice are characterized by the absence of inter-calated cell markers including AE4. To test whether Slc4a9 is a direct target gene of Foxi1, an AE4 promoter construct was generated for a cell-based reporter gene assay. Co-transfection with the Foxi1 cDNA resulted in an approx. 100-fold activation of the AE4 promoter construct. By truncating the AE4 promoter at the 5′-end, we demonstrate that a fragment of approx. 462 bp upstream of the transcription start point is sufficient to mediate activation by Foxi1. Sequence analysis of this region revealed at least eight potential binding sites for Foxi1 in both sense and antisense orientation. Only one element was bound by recombinant Foxi1 protein in bandshift assays. Mutation of this site abolished both binding in bandshift assays and transcriptional activation by co-transfection of Foxi1 in the reporter gene assay. We thus identify the AE4 promoter as a direct target of Foxi1.
Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability
Authors: Galán María , Vincent Olivier , Roncero Isabel , Azriel Sharona , Boix-Pallares Pedro , Delgado-Alvarez Elías , Díaz-Cadórniga Francisco , Blázquez Enrique , Navas María-Angeles
Published: December 2005

Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the β-cell. Heterozygous mutations in the glucokinase-encoding GCK gene, which result in a reduction of the enzymatic activity, cause the monogenic form of diabetes, MODY2 (maturity-onset diabetes of the young 2). We have identified and functionally characterized missense mutations in the GCK gene in diabetic families that result in protein mutations Leu165→Phe, Glu265→Lys and Thr206→Met. The first two are novel GCK mutations that co-segregate with the diabetes phenotype in their respective families and are not found in more than 50 healthy control individuals. In order to measure the biochemical effects of these missense mutations on glucokinase activity, we bacterially expressed and affinity-purified islet human glucokinase proteins carrying the respective mutations and fused to GST (glutathione S-transferase). Enzymatic assays on the recombinant proteins revealed that mutations Thr206→Met and Leu165→Phe strongly affect the kinetic parameters of glucokinase, in agreement with the localization of both residues close to the active site of the enzyme. In contrast, mutation Glu265→Lys, which has a weaker effect on the kinetics of glucokinase, strongly affects the protein stability, suggesting a possible structural defect of this mutant protein. Finally, none of the mutations tested appears to affect the interaction of gluco-kinase with the glucokinase regulatory protein in the yeast two-hybrid system.
Expression and characterization of a histidine-rich protein, Hpn: potential for Ni2+ storage in Helicobacter pylori
Authors: Ge Ruiguang , Watt Rory M. , Sun Xuesong , Tanner Julian A. , He Qing-Yu , Huang Jian-Dong , Sun Hongzhe
Published: December 2005

Hpn is a small cytoplasmic protein found in Helicobacter pylori, which binds Ni2+ ions with moderate affinity. Consisting of 60 amino acids, the protein is rich in histidine (28 residues, 46.7%), as well as glutamate, glycine and serine residues (in total 31.7%), and contains short repeating motifs. In the present study, we report the detailed biophysical characterization of the multimeric status and Ni2+-binding properties of purified recombinant Hpn under physiologically relevant conditions. The protein exists as an equilibration of multimeric forms in solution, with 20-mers (approx. 136 kDa) being the predominant species. Using equilibrium dialysis, ICP-MS (inductively coupled plasma MS) and UV/visible spectroscopy, Hpn was found to bind five Ni2+ ions per monomer at pH 7.4, with a dissociation constant (Kd) of 7.1 μM. Importantly, Ni2+ binding to Hpn is reversible: metal is released either in the presence of a chelating ligand such as EDTA, or at a slightly acidic pH (pH for half dissociation, pH1/2 ∼6.3). Ni2+ binding induces conformational changes within the protein, increasing β-sheet and reducing α-helical content, from 22% to 37%, and 20% to 10% respectively. Growth curves of Escherichia coli BL21(DE3) both with and without the hpn gene performed under Ni2+ pressure clearly implied a role for Hpn to protect the cells from higher concentrations of external metal ions. Similarly, the accumulation of Ni2+ in these cells expressing Hpn from a plasmid was approx. 4-fold higher than in uninduced controls or control cultures that lacked the plasmid. Similarly, levels of Ni2+ in wild-type H. pylori 26695 cells were higher than those in H. pylori hpn-deletion mutant strains. Hpn may potentially serve multiple roles inside the bacterium: storage of Ni2+ ions in a ‘reservoir’; donation of Ni2+ to other proteins; and detoxification via sequestration of excess Ni2+.
LOX-1 scavenger receptor mediates calcium-dependent recognition of phosphatidylserine and apoptotic cells
Authors: Murphy Jane E. , Tacon Daryl , Tedbury Philip R. , Hadden Jonathan M. , Knowling Stuart , Sawamura Tatsuya , Peckham Michelle , Phillips Simon E. V. , Walker John H. , Ponnambalam Sreenivasan
Published: December 2005

The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) scavenger receptor regulates vascular responses to oxidized-low-density-lipoprotein particles implicated in atherosclerotic plaque formation. LOX-1 is closely related to C-type lectins, but the mechanism of ligand recognition is not known. Here we show that human LOX-1 recognizes a key cellular phospholipid, PS (phosphatidylserine), in a Ca2+-dependent manner, both in vitro and in cultured cells. A recombinant, folded and glycosylated LOX-1 molecule binds PS, but not other phospholipids. LOX-1 recognition of PS was maximal in the presence of millimolar Ca2+ levels. Mg2+ was unable to substitute for Ca2+ in LOX-1 binding to PS, indicating a Ca2+-specific requirement for bivalent cations. LOX-1-mediated recognition of PS-containing apoptotic bodies was dependent on Ca2+ and was decreased to background levels by bivalent-cation chelation, LOX-1-blocking antibodies or PS-containing liposomes. The LOX-1 membrane protein is thus a Ca2+-dependent phospholipid receptor, revealing novel recognition of phospholipids by mammalian lectins.
Fission yeast Dss1 associates with the proteasome and is required for efficient ubiquitin-dependent proteolysis
Authors: Jossé Lyne , Harley Margaret E. , Pires Isabel M. S. , Hughes David A.
Published: December 2005

Human DSS1 associates with BRCA2, a tumour suppressor protein required for efficient recombinational DNA repair, but the biochemical function of DSS1 is not known. Orthologues of DSS1 are found in organisms such as budding yeast and fission yeast that do not have BRCA2-related proteins, indicating that DSS1 has a physiological role independent of BRCA2. The DSS1 orthologue in Saccharomyces cerevisiae has been shown to associate with the 26 S proteasome and, in the present paper, we report that in the distantly related fission yeast Schizosaccharomyces pombe, Dss1 associates with the 19 S RP (regulatory particle) of the 26 S proteasome. A role for S. pombe Dss1 in proteasome function is supported by three lines of evidence. First, overexpression of two components of the 19 S RP, namely Pad1/Rpn11 and Mts3/Rpn12, rescued the temperature-sensitive growth defect of the dss1 mutant. Secondly, the dss1 mutant showed phenotypes indicative of a defect in proteasome function: growth of the dss1 mutant was inhibited by low concentrations of L-canavanine, an amino acid analogue, and cells of the dss1 mutant accumulated high molecular mass poly-ubiquitylated proteins. Thirdly, synthetic growth defects were found when the dss1 mutation was combined with mutations in other proteasome subunit genes. These findings show that DSS1 has an evolutionarily conserved role as a regulator of proteasome function and suggest that DSS1 may provide a link between BRCA2 and ubiquitin-mediated proteolysis in human cells.
Intracellular signalling pathways activated by leptin
Authors: Frühbeck Gema
Published: December 2005

Leptin is a versatile 16 kDa peptide hormone, with a tertiary structure resembling that of members of the long-chain helical cytokine family. It is mainly produced by adipocytes in proportion to fat size stores, and was originally thought to act only as a satiety factor. However, the ubiquitous distribution of OB-R leptin receptors in almost all tissues underlies the pleiotropism of leptin. OB-Rs belong to the class I cytokine receptor family, which is known to act through JAKs (Janus kinases) and STATs (signal transducers and activators of transcription). The OB-R gene is alternatively spliced to produce at least five isoforms. The full-length isoform, OB-Rb, contains intracellular motifs required for activation of the JAK/STAT signal transduction pathway, and is considered to be the functional receptor. Considerable evidence for systemic effects of leptin on body mass control, reproduction, angiogenesis, immunity, wound healing, bone remodelling and cardiovascular function, as well as on specific metabolic pathways, indicates that leptin operates both directly and indirectly to orchestrate complex pathophysiological processes. Consistent with leptin's pleiotropic role, its participation in and crosstalk with some of the main signalling pathways, including those involving insulin receptor substrates, phosphoinositide 3-kinase, protein kinase B, protein kinase C, extracellular-signal-regulated kinase, mitogen-activated protein kinases, phosphodiesterase, phospholipase C and nitric oxide, has been observed. The impact of leptin on several equally relevant signalling pathways extends also to Rho family GTPases in relation to the actin cytoskeleton, production of reactive oxygen species, stimulation of prostaglandins, binding to diacylglycerol kinase and catecholamine secretion, among others.
SLPI and elafin: one glove, many fingers
Authors: Williams Steven E. , Brown Thomas I. , Roghanian Ali , Sallenave Jean-Michel
Published: December 2005

Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.
Short interfering RNA (siRNA): tool or therapeutic?
Authors: Cejka Daniel , Losert Doris , Wacheck Volker
Published: December 2005

Gene silencing by siRNA (short interfering RNA) is a still developing field in biology and has evolved as a novel post-transcriptional gene silencing strategy with therapeutic potential. With siRNAs, virtually every gene in the human genome contributing to a disease becomes amenable to regulation, thus opening unprecedented opportunities for drug discovery. Besides the well-established role for siRNA as a tool for target screening and validation in vitro, recent progress of siRNA delivery in vivo raised expectations for siRNA drugs as the up-and-coming ‘magic bullet’. Whether siRNA compounds will make it as novel chemical entities from ‘bench to bedside’ will probably depend largely on improving their pharmacokinetics in terms of plasma stability and cellular uptake. Whereas locally administered siRNAs have already entered the first clinical trials, strategies for successful systemic delivery of siRNA are still in a preclinical stage of development. Irrespective of its therapeutic potential, RNAi (RNA interference) has unambiguously become a valuable tool for basic research in biology and thereby it will continue to have a major impact on medical science. In this review, we will give a brief overview about the history and current understanding of RNAi and focus on potential applications, especially as a therapeutic option to treat human disease.