bluuebunny/crossref_metadata_2025 huggingface dataset:
SIERRA observations of Alfvénic processes in the topside auroral ionosphere
Authors: Klatt E. M. , Kintner P. M. , Seyler C. E. , Liu K. , MacDonald E. A. , Lynch K. A.
Published: August 2005

The NASA SIERRA rocket mission was a multiple‐payload, auroral plasma physics experiment that flew into a 100 nT auroral substorm at altitudes up to 735 km over the Poker Flat Research Range in Alaska on 14 January 2002. The flight environment was composed of an equatorward region of inverted‐V electron precipitation followed by a poleward region of mostly field‐aligned suprathermal electron bursts. In the inverted‐V region, the measured δE/δB implied a stationary field‐aligned current configuration. In the poleward region, the average electric field was much larger, 50 mV/m compared with 20 mV/m, included time‐varying components that exceeded 100 mV/m with periods of 0.5–5 s, and δE/δB was Alfvénic. Our multiple‐payload observations show that the perpendicular scale size of most of these waves is on the order of a kilometer or less. At smaller scales, broadband, extremely low‐frequency (BBELF) fluctuations associated with observed Alfvén waves had spectral ratios (δE/δB) appropriate for inertial Alfvén waves larger and smaller than the electron inertial length (λe = c/ωpe), but the phase angle differences between δE and δB indicate mostly propagating Alfvén waves for spatial scales much larger than λe and a transition to spatial perturbations, embedded in the plasma, for scales near λe. The data are consistent with the model of kilometer‐scale oblique Alfvénic arcs that are unstable to the current shear‐driven instabilities discussed in previous theoretical investigations, lending support to the conclusion that the observed BBELF fluctuations are likely driven by the wave‐associated field‐aligned current shear.
Ring current and the magnetosphere‐ionosphere coupling during the superstorm of 20 November 2003
Authors: Ebihara Y. , Fok M.‐C. , Sazykin S. , Thomsen M. F. , Hairston M. R. , Evans D. S. , Rich F. J. , Ejiri M.
Published: August 2005

We investigated the impact on the terrestrial ring current of a coronal mass ejection (CME) and the associated magnetic cloud that severely disturbed the Earth's magnetosphere on 20 November 2003. This CME decreased the Dst index to −472 nT, which makes it the second largest storm, based on the minimum Dst index values, observed between 1957 and 2004. Data from the DMSP, NOAA, and LANL satellites showed the unique characteristics of this storm; a polar cap potential that increased to at least 200 kV, a polar cap boundary that moved as low as about 60° MLAT, a plasma sheet density that increased to 5 cm−3 at L = 6.6 when the Dst index was near its minimum, and the inner edge of the plasma sheet ion population that penetrated into a region for which L ≤ 1.5. In order to study the dynamics of the ring current and the associated magnetosphere‐ionosphere coupling, we performed a ring current simulation that computed the evolution of the phase space density of the ring current ions and the closure of the electric current between the magnetosphere and the ionosphere. Major results were as follows: (1) The ring current, in terms of the Dst index and the inner edge of the plasma sheet, can result from the enhancement of the convection electric field, given the polar cap potentials used in the model; (2) The solar wind particles probably penetrated quickly into the geosynchronous altitude on the nightside with a lag of about 80 min, resulting in further enhancement of the ring current; (3) Dense geocoronal neutral hydrogen or a large coefficient of pitch angle diffusion (>10−4 s−1) is probably needed to account for the rapid motion of the inner edge of the plasma sheet (or the ring current) population to a higher L value; (4) Both the simulated and observed field‐aligned current (FAC) distributions show multiple current sheets, rather than the normally expected two current sheets. Fluctuations in the polar cap potential and the plasma sheet density are believed to cause the multiple sheets of field‐aligned currents; (5) The equatorward edge of the Region 2 type field‐aligned currents was observed to expand as low as 40° MLAT, which is consistent with the simulation; and (6) The convection pattern can be much more complicated than an average one due to a strong Region 2 FAC. A noticeable feature was the reversal of the zonal ionospheric plasma flow that emerged on the dawnside. In particular, a westward flow was observed in the equatorial region of the eastward plasma flow at dawn. Its speed had a local maximum of about 5° equatorward of the flow reversal. The flow reversal is thought to have resulted from the relatively strong shielding electric field.
Low‐frequency plasma oscillations at Mars during the October 2003 solar storm
Authors: Espley J. R. , Cloutier P. A. , Crider D. H. , Brain D. A. , Acuña M. H.
Published: August 2005

The powerful x‐class flare which occurred on the Sun on 28 October 2003 had important effects on plasma environments throughout the solar system. We present here observations of the effects at Mars from the Mars Global Surveyor (MGS) Magnetometer/Electron Reflectometer experiment. In particular we focus on the changes in the nature of the magnetic oscillations observed at an altitude of 400 km (MGS's current orbital altitude) during the passage of the solar storm. We find that strong, regular oscillations are observed in both the B∥ and B⊥ components of the magnetic field at all solar zenith angles. We emphasize in particular the powerful, coherent oscillations observed in the normally quiet nightside region. These oscillations carry power at the proton gyrofrequency and at and below the oxygen gyrofrequency. This implies that ions of planetary origin are interacting with the solar wind plasma and raises the possibility that significant atmospheric loss may occur during the passage of large solar storms at Mars.
Nonlinear decay of foreshock Langmuir waves in the presence of plasma inhomogeneities: Theory and Cluster observations
Authors: Soucek J. , Krasnoselskikh V. , Dudok de Wit T. , Pickett J. , Kletzing C.
Published: August 2005

The intense high‐frequency electrostatic waves observed in the terrestrial foreshock often have a form of a superposition of two monochromatic waves close to the plasma frequency. We suggest an interpretation of these spectra as signatures of nonlinear decay of Langmuir waves to electron‐sound and ion‐sound secondary waves. This decay instability is known to have different properties in inhomogeneous plasma, namely the threshold amplitude of this instability is inversely proportional to the scale of the inhomogeneity. We show that the observed dependence of the wave amplitude on the modulation scale of the wave packets is consistent with this property and the theory of absolute decay instability in inhomogeneous plasma can be applied to explain the satellite observations. In this study we used electric field data from the Wide Band Data instrument on board Cluster satellites.
Autocatalytic water vapor production as a source of large mixing ratios within the middle to upper mesosphere
Authors: Sonnemann G. R. , Grygalashvyly M. , Berger U.
Published: August 2005

Different measurements have shown that large water vapor mixing ratios occur in the upper mesosphere in high‐latitude summer just under conditions of strongest solar insolation where photolysis should effectively reduce its value. The analysis of this finding by means of a high‐resolution one‐dimensional model supplies evidence that water vapor is autocatalytically formed below a crossover height of about 65 km and destroyed by photolysis above this altitude. We found three catalytic cycles of water vapor formation from the molecular hydrogen reservoir. Hydrogen radicals, resulting for the most part from water vapor by photolysis and oxidation by O(1D), act as catalysts. A great number of the hydrogen radicals return to water vapor, particularly under the condition of small atomic hydrogen concentration decreasing strongly with decreasing height. In the upper domain under the condition of large atomic hydrogen concentrations the formation of molecular hydrogen will be favored. Favorable conditions for large water vapor mixing ratios at high altitudes are given by an accelerated upward vertical wind. Below the crossover level the vertical wind should be weak so that H2 and CH4 have sufficient time to oxidize into H2O, a very slow process. Above this level, however, the vertical wind should be strong in order to lift the air, enriched with H2O, as quickly as possible to greater heights before the dissociation has an effective impact on the H2O distribution. Such accelerated upward vertical winds are typical of the mesosphere under certain seasonally latitudinal conditions. Calculations by means of our three‐dimensional model COMMA‐IAP yield seasonal‐latitudinal patterns and variations approximately comparable with those of the HALOE observations. In the paper we discuss the chemical background and the interplay between dynamics and chemistry in more detail.
Absence of isotope exchange in the reaction of N2O + O(1D) and the global Δ17O budget of nitrous oxide
Authors: Kaiser Jan , Röckmann Thomas
Published: August 2005

Yung et al. (2004) suggested that N2O exchanges isotopes with O(1D), analogous to CO2, which would represent an additional contribution to the 17O isotope anomaly of atmospheric N2O. Here we demonstrate by isotope measurements of N2O remaining after partial conversion with O(1D) that this isotope exchange most likely proceeds at <1% of the total N2O + O(1D) reaction rate. It can therefore be neglected as a source of the 17O anomaly of atmospheric N2O, corroborating arguments based on previous indirect experimental evidence of the absence of such isotope exchange (Röckmann and Kaiser, 2005). In addition, we present a balanced budget of the 17O anomaly of atmospheric N2O, based on N2O sources at Earth's surface and in the atmosphere: the reactions of N2 + O(1D) + M, NH2 + NO2, and N + NO2, industrial processes, biomass burning, and the “numerical” source from microbial nitrogen conversion reactions (Kaiser et al., 2004b).
Global response of the low‐latitude to midlatitude ionosphere due to the Bastille Day flare
Authors: Huba J. D. , Warren H. P. , Joyce G. , Pi X. , Iijima B. , Coker C.
Published: August 2005

The first global simulation study and comparison to data of the ionospheric effects associated with the enhanced EUV irradiance of the Bastille Day flare are presented. This is done by incorporating a time‐dependent EUV spectrum, based on data and hydrodynamic modeling, into the NRL ionosphere model SAMI3. The simulation results indicate that the total electron content (TEC) increases to over 7 TEC units in the daytime, low‐latitude ionosphere. In addition, it is predicted that the maximum density in the F‐layer (NmF2) increases by ≲20% and that the height of the maximum electron density (HmF2) decreases by ≲20%. These results are explained by the increased ionization at altitudes <400 km which increases TEC and NmF2 while decreasing HmF2. The results are in reasonably good agreement with data obtained from GPS satellites and the TOPEX satellite.
Variable seasonal coupling between air and ground temperatures: A simple representation in terms of subsurface thermal diffusivity
Authors: Pollack Henry N. , Smerdon Jason E. , van Keken Peter E.
Published: August 2005

The utility of subsurface temperatures as indicators of temperature changes at Earth's surface rests upon an assumption of strong coupling between surface air temperature (SAT) and ground surface temperature (GST). Here we describe a simple representation of this coupling in terms of a variable thermal diffusivity in the upper meter of the subsurface. The variability is tied to daily SAT, precipitation, and snow cover, but does not incorporate the physical details of these and the many other factors that influence the air‐ground interface in many high‐fidelity land‐surface models. Our simple model reduces the difference between observed and modeled temperatures by a factor of 3 to 4 over a model with uniform diffusivity driven only by SAT. This simple representation of air‐ground coupling offers a means of simulating subsurface temperatures using only archived meteorological records and creates the potential for examining the long term character of air‐ground temperature coupling.
Parkinson's Disease and Brain Mitochondrial Dysfunction: A Functional Phosphorus Magnetic Resonance Spectroscopy Study
Authors: Rango Mario , Bonifati Cristiana , Bresolin Nereo
Published: August 2005

In spite of several evidences for a mitochondrial impairment in Parkinson's disease (PD), so far it has not been possible to show in vivo mitochondrial dysfunction in the human brain of PD patients. The authors used the high temporal and spatial resolution 31 phosphorus magnetic resonance spectroscopy (31P MRS) technique, which they have previously developed in normal subjects and in patients with mitochondrial diseases to study mitochondrial function by observing high-energy phosphates (HEPs) and intracellular pH (pH) in the visual cortex of 20 patients with PD and 20 normal subjects at rest, during, and after visual activation. In normal subjects, HEPs remained unchanged during activation, but rose significantly (by 16%) during recovery, and pH increased during visual activation with a slow return to rest values. In PD patients, HEPs were within the normal range at rest and did not change during activation, but fell significantly (by 36%) in the recovery period; pH did not reveal a homogeneous pattern with a wide spread of values. Energy unbalance under increased oxidative metabolism requirements, that is, the postactivation phase, discloses a mitochondrial dysfunction that is present in the brain of patients with PD even in the absence of overt clinical manifestations, as in the visual cortex. This is in agreement with our previous findings in patients with mitochondrial disease without clinical central nervous system (CNS) involvement. The heterogeneity of the physicochemical environment (i.e., pH) suggests various degrees of subclinical brain involvement in PD. The combined use of MRS and brain activation is fundamental for the study of brain energetics in patients with PD and may prove an important tool for diagnostic purposes and, possibly, to monitor therapeutic interventions.
Increased Infarct Size and Lack of Hyperphagic Response after Focal Cerebral Ischemia in Peroxisome Proliferator-Activated Receptor β-Deficient Mice
Authors: Arsenijevic Denis , de Bilbao Fabienne , Plamondon Julie , Paradis Eric , Vallet Philippe , Richard Denis , Langhans Wolfgang , Giannakopoulos Panteleimon
Published: August 2005

Peroxisome proliferator-activated receptors (PPARs) are involved in energy expenditure, regulation of inflammatory processes, and cellular protection in peripheral tissues. Among the different types of PPARs, PPARβ is the only one to be widely expressed in cortical neurons. Using PPARβ knockout (KO) mice, we report here a detailed investigation of the role of PPARβ in cerebral ischemic damage, associated inflammatory and antioxidant processes as well as food intake regulation after middle cerebral artery occlusion (MCAO). The PPARβ KO mice had a two-fold increase in infarct size compared with wild-type (WT) mice. Brain oxidative stress was dramatically enhanced in these KO mice, as documented by an increased content of malondialdehyde, decreased levels of glutathione and manganese superoxide dismutase, and no induction of uncoupling protein 2 (UCP2) mRNA. Unlike WT mice, PPARβ KO mice showed a marked increase of prooxidant interferon-gamma but no induction of nerve growth factor and tumor necrosis factor alpha after MCAO. In WT mice, MCAO resulted in inflammation-specific transient hyperphagia from day 3 to day 5 after ischemia, which was associated with an increase in neuropeptide Y (NPY) mRNA. This hyperphagic phase and NPY mRNA induction were not observed in PPARβ KO mice. Furthermore, our study also suggests for the first time that UCP2 is involved in MCAO food intake response. These data indicate that PPARβ plays an important role in integrating and regulating central inflammation, antioxidant mechanisms, and food intake after MCAO, and suggest that the use of PPARβ agonists may be of interest for the prevention of central ischemic damage.
CuZn-SOD Deficiency, Rather than Overexpression, is Associated with Enhanced Recovery and Attenuated Activation of NF-κB After Brain Trauma in Mice
Authors: Beni Sara M , Tsenter Jeanna , Alexandrovich Alexander G , Galron-Krool Nili , Barzilai Ari , Kohen Ron , Grigoriadis Nikolas , Simeonidou Constantina , Shohami Esther
Published: August 2005

Superoxide-dismutases (SOD) catalyze O2− conversion to hydrogen peroxide (H2O2) and with other antioxidant enzymes and low molecular weight antioxidants (LMWA) constitute endogenous defense mechanisms. We first assessed the effects of SOD1 levels on outcome after closed head injury (CHI) and later, based on these results, the effects of SOD−/- deficiency on cellular redox homeostasis. Superoxide-dismutase 1-deficient (SOD1−/-) and -overexpressing (transgenic (Tg)) mice and matched wild-type (WT) controls were subjected to CHI and outcome (neurobehavioral and memory functions) was assessed during 14 days. Brain edema, LMWA, and SOD2 activity were measured along with histopathological analysis. Transactivation of nuclear factor-kappa B (NF- κB) was evaluated by electromobility shift assay. Mortality, motor, and cognitive outcome of Tg and WT mice were comparable. Mortality and edema were similar in SOD1−/- and WT mice, yet, unexpectedly, SOD1−/- displayed better neurobehavioral recovery ( P < 0.05) at 14 days after CHI. Basal LMWA were higher in the cortex and liver of SOD1−/- mice ( P < 0.05) and similar to WT in the cerebellum. Five minutes after CHI, cortical LMWA decreased only in SOD1−/- mice. One week after CHI, SOD2 activity decreased fourfold in WT cortex ( P < 0.001), but was preserved in the SOD1−/- . Constitutive NF- κB transactivation was comparably low in SOD1−/- and WT; however, CHI induced a robust NF- κB activation that was absent in SOD1−/- cortices ( P > 0.005 versus WT). At the same time, immunohistochemical analysis of brain sections revealed that astrogliosis and neurodegeneration were of lesser severity in SOD1−/- mice. We suggest that SOD1 deficiency impairs H2O2-mediated activation of NF- κB, decreasing death-promoting signals, and leading to better outcome.
Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter
Authors: Nyga Rémy , Pecquet Christian , Harir Noria , Gu Haihua , Dhennin-Duthille Isabelle , Régnier Aline , Gouilleux-Gruart Valérie , Lassoued Kaïss , Gouilleux Fabrice
Published: August 2005

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel–JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.
External mechanical force as an inhibition process in kinesin's motion
Authors: Ciudad Aleix , Sancho José María
Published: August 2005

We analysed published force–velocity data for kinesin using classical Michaelis–Menten kinetic theory and found that the effect of force on the stepping rate of kinesin is analogous to the effect of a mixed inhibitor in classical inhibition theory. We derived an analytical expression for the velocity of kinesin (the stepping rate, equal to the ATP turnover rate) as a function of ATP concentration and force, and showed that it accurately predicts the observed single molecule stepping rate of kinesin under a variety of conditions.
Identification of a functional hypoxia-responsive element that regulates the expression of the egl nine homologue 3 (egln3/phd3) gene
Authors: Pescador Nuria , Cuevas Yolanda , Naranjo Salvador , Alcaide Marisa , Villar Diego , Landázuri Manuel O. , del Peso Luis
Published: August 2005

Low oxygen levels induce an adaptive response in cells through the activation of HIFs (hypoxia-inducible factors). These transcription factors are mainly regulated by a group of proline hydroxylases that, in the presence of oxygen, target HIF for degradation. The expression of two such enzymes, EGLN1 [EGL nine homologous protein 1, where EGL stands for egg laying defective (Caenorhabditis elegans gene)] and EGLN3, is induced by hypoxia through a negative feedback loop, and we have demonstrated recently that hypoxic induction of EGLN expression is HIF-dependent. In the present study, we have identified an HRE (hypoxia response element) in the region of the EGLN3 gene using a combination of bioinformatics and biological approaches. Initially, we isolated a number of HRE consensus sequences in a region of 40 kb around the human EGLN3 gene and studied their evolutionary conservation. Subsequently, we examined the functionality of the conserved HRE sequences in reporter and chromatin precipitation assays. One of the HREs, located within a conserved region of the first intron of the EGLN3 gene 12 kb downstream of the transcription initiation site, bound HIF in vivo. Furthermore, this sequence was able to drive reporter gene expression under conditions of hypoxia in an HRE-dependent manner. Indeed, we were able to demonstrate that HIF was necessary and sufficient to induce gene expression from this enhancer sequence.
Controlled alteration of the shape and conformational stability of α-helical cell-lytic peptides: effect on mode of action and cell specificity
Authors: Zelezetsky Igor , Pacor Sabrina , Pag Ulrike , Papo Niv , Shai Yechiel , Sahl Hans-Georg , Tossi Alessandro
Published: August 2005

A novel method, based on the rational and systematic modulation of macroscopic structural characteristics on a template originating from a large number of natural, cell-lytic, amphipathic α-helical peptides, was used to probe how the depths and shapes of hydrophobic and polar faces and the conformational stability affect antimicrobial activity and selectivity with respect to eukaryotic cells. A plausible mode of action explaining the peptides' behaviour in model membranes, bacteria and host cells is proposed. Cytotoxic activity, in general, correlated strongly with the hydrophobic sector depth, and required a majority of aliphatic residue side chains having more than two carbon atoms. It also correlated significantly with the size of polar sector residues, which determines the penetration depth of the peptide via the so-called snorkel effect. Both an oblique gradient of long to short aliphatic residues along the hydrophobic face and a stabilized helical structure increased activity against host cells but not against bacteria, as revealed by haemolysis, flow cytofluorimetric studies on lymphocytes and surface plasmon resonance studies with model phosphatidylcholine/cholesterol membranes. The mode of interaction changes radically for a peptide with a stable, preformed helical conformation compared with others that form a structure only on membrane binding. The close correlation between effects observed in biological and model systems suggests that the ‘carpet model’ correctly represents the type of peptides that are bacteria-selective, whereas the behaviour of those that lyse host cells is more complex.
An ordered sequential mechanism for Factor IX and Factor IXa binding to platelet receptors in the assembly of the Factor X-activating complex
Authors: Yang Xia , Walsh Peter N.
Published: August 2005

To define the contributions of the Ω-loop of the Gla (γ-carboxyglutamic acid) domain and the EGF2 (second epidermal growth factor) domain of FIXa (Factor IXa) in the assembly of the FX-activating complex on activated platelets and phospholipid membranes, three recombinant FIXa chimeras were prepared with corresponding residues from the homologous coagulation protein, FVII: (i) Gly4–Gln11 (FIXa7Ωloop), (ii) Cys88–Cys124 (FIXa7EGF2), and (iii) both Gly4–Gln11 and Cys88–Cys124 (FIXa7Ωloop7EGF2). All three chimeras were similar to wild-type FIXa, as assessed by SDS/PAGE, active-site titration, content of Gla residues, activation rates by FXIa and rates of FXa generation in solution. Titrations of FX or FVIIIa on SFLLRN peptide-activated platelets and on phospholipid vesicles in the presence of FVIIIa revealed normal substrate and cofactor binding to all chimeras. In kinetic assays in the presence of phospholipid vesicles and FVIIIa, compared with wild-type FIXa Kd, app∼4 nM, the FIX7Ωloop chimera showed a 1.6-fold increase in Kd, app, the FIX7EGF2 chimera had a 7.4-fold increase in Kd, app, and the FIX7Ωloop7EGF2 chimera showed a 21-fold increase in Kd, app. In kinetic assays and equilibrium platelet-binding assays with activated platelets and FVIIIa, compared with wild-type FIXa (Vmax∼5 nM min−1; Kd, app∼0.5 nM; Bmax∼550 sites/platelet; Kd∼0.5 nM), the FIX7Ωloop chimera displayed 2-fold decreases in Vmax and Bmax and 2-fold increases in Kd, app and Kd. The FIX7EGF2 chimera displayed 2-fold decreases in Vmax and Bmax and 10-fold increases in Kd, app and Kd. The FIX7Ωloop7EGF2 chimera showed non-saturable curves and severely impaired rates of FXa generation, and non-saturable, non-specific, low-level binding to activated platelets. Thus both the Gla domain Ω-loop (Gly4–Gln11) and the EGF2 domain (Cys88–Cys124) are required to mediate the normal assembly of the FX-activating complex on activated platelets and on phospholipid membranes.
Site-specific base changes in the coding or promoter region of the human β- and γ-globin genes by single-stranded oligonucleotides
Authors: Yin Wenxuan , Kren Betsy T. , Steer Clifford J.
Published: August 2005

SSOs (single-stranded oligonucleotides) can mediate site-specific alteration of base-pairs in episomal and chromosomal target genes in mammalian cells. The TNE (targeted nucleotide exchange) can result in either repair or mutation of a gene sequence and is mediated through endogenous DNA repair pathway(s). Thus the approach provides a technique for the treatment of monogenic disorders associated with specific point mutations such as SCD (sickle cell disease). We studied the potential application of SSOs for SCD by introducing either an A to T substitution at the sixth codon of the human β-globin gene (sickle locus) or a C to G mutation at −202 of the Gγ-globin gene promoter region. The latter TNE is an alternative strategy to ameliorate the clinical manifestations of sickle cell anaemia by re-activating fetal haemoglobin gene expression in adult erythrocytes. A sensitive and valid PCR assay system was developed, which allows detection of point mutations as low as 0.01% at these sites. Using this system, TNE between 0.01 and 0.1% at the sickle locus or γ-globin gene promoter region was detected after transfection with SSOs in cultured human cell lines. TNE in the Gγ-globin promoter region exhibited varying degrees of strand bias that was dependent on SSO design and the cell's DNA mismatch repair activity. The results suggest that the endogenous DNA repair machinery may permit SSO correction of the sickle defect by modification of the β- and/or γ-globin genes.
RhoGDI: multiple functions in the regulation of Rho family GTPase activities
Authors: Dovas Athanassios , Couchman John R.
Published: August 2005

RhoGDI (Rho GDP-dissociation inhibitor) was identified as a down-regulator of Rho family GTPases typified by its ability to prevent nucleotide exchange and membrane association. Structural studies on GTPase–RhoGDI complexes, in combination with biochemical and cell biological results, have provided insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association–dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability and the importance of the particular membrane microenvironment that represents the site of action for GTPases. All these results point to a wider role for RhoGDI than initially perceived, making it a binding partner that can tightly control Rho GTPases, but which also allows them to reach their full spectrum of activities.
Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor
Authors: Lanctot Pascal M. , Leclerc Patrice C. , Clément Martin , Auger-Messier Mannix , Escher Emanuel , Leduc Richard , Guillemette Gaétan
Published: August 2005

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT1 receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.
Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast
Authors: Liao Xin-Hua , Zhang Ming-Liang , Yang Cui-Ping , Xu Lu-Xia , Zhou Jin-Qiu
Published: August 2005

Telomerase is a cellular reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. In Saccharomyces cerevisiae, Est2p (telomerase catalytic subunit) and Tlc1 (telomerase RNA template subunit) constitute the telomerase core complex. We co-overexpressed GST (glutathione S-transferase)–Est2p and Tlc1 in S. cerevisiae, and reconstituted the telomerase activity. The GST–Est2p–Tlc1 complex was partially purified by ammonium sulphate fractionation and affinity chromatography on glutathione beads, and the partially purified telomerase did not contain the other two subunits of the telomerase holoenzyme, Est1p and Est3p. The purified recombinant GST–Est2p–Tlc1 telomerase core complex could specifically add nucleotides on to the single-stranded TG1–3 primer in a processive manner, but could not translocate to synthesize more than one telomeric repeat. The purified telomerase core complex exhibited different activities when primers were paired with the Tlc1 template at different positions. The procedure of reconstitution and purification of telomerase core enzyme that we have developed now allows for further mechanistic studies of the functions of other subunits of the telomerase holoenzyme as well as other telomerase regulation proteins.
Cecropin P1 and novel nematode cecropins: a bacteria-inducible antimicrobial peptide family in the nematode Ascaris suum
Authors: Pillai Ajitha , Ueno Satoshi , Zhang Hong , Lee Jae Min , Kato Yusuke
Published: August 2005

Cecropin P1 was first identified as a mammalian antimicrobial peptide isolated from the pig intestine. Much research aimed at characterizing this peptide has been reported. Recently, the workers who discovered the peptide corrected their original conclusion, and confirmed that this peptide originates in fact from the pig intestinal parasitic nematode, Ascaris suum. In the present study, we carried out a semi-exhaustive search for bacteria-inducible transcripts in A. suum by the cDNA subtraction method. The transcripts encoding cecropin P1 and novel Ascaris cecropins, designated cecropins P2, P3 and P4, were found to be positively induced factors. Chemically synthesized Ascaris cecropins were bactericidal against a wide range of microbes, i.e. Gram-positive (Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus) and Gram-negative (Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens and Esherichia coli) bacteria, and were weakly but detectably active against yeasts (Saccharomyces cerevisiae and Candida albicans). Cecropin P1-like sequences were also detected at least in two other species (Ascaris lumbricoides and Toxocara canis) of the Ascarididae. All Ascaris cecropin precursors contain an acidic pro-region connected by a tetra-basic cleavage site at the C-terminus. Such an acidic pro-region is also reported to be present in the tunicate cecropin-type antimicrobial peptide styelin. On the basis of the evolutionary position of nematodes and tunicates, the ancestral cecropin may have contained the acidic pro-region at the C-terminus.
Surface salt bridges modulate the DNA site size of bacterial histone-like HU proteins
Authors: Kamau Edwin , Tsihlis Nick D. , Simmons L. Alice , Grove Anne
Published: August 2005

Bacterial histone-like DNA-binding proteins are best known for their role in compacting the genomic DNA. Of these proteins, HU is ubiquitous and highly conserved across the eubacterial kingdom. Using the HBsu (Bacillus subtilis-encoded HU homologue) as a model, we explore here the molecular basis for the ability of some HU homologues to engage a longer approx. 35 bp DNA site as opposed to the much shorter sites reported for other homologues. Using electrophoretic mobility-shift assays, we show that the DNA site size for HBsu is approx. 10–13 bp and that a specific surface salt bridge limits the DNA site size for HBsu. Surface exposure of the highly conserved Lys3, achieved by substitution of its salt-bridging partner Asp26 with Ala, leads to enhanced DNA compaction by HBsu-D26A (where D26A stands for the mutant Asp26→Ala), consistent with the interaction of Lys3 with the ends of a 25 bp duplex. Both HBsu and HBsu-D26A bend DNA, as demonstrated by their equivalent ability to promote ligase-mediated DNA cyclization, indicating that residues involved in mediating DNA kinks are unaltered in the mutant protein. We suggest that Lys3 is important for DNA wrapping due to its position at a distance from the DNA kinks where it can exert optimal leverage on flanking DNA and that participation of Lys3 in a surface salt bridge competes for its interaction with DNA phosphates, thereby reducing the occluded site size.
Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines
Authors: Sandy James , Mushtaq Adeel , Holton Simon J. , Schartau Pamela , Noble Martin E. M. , Sim Edith
Published: August 2005

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.
Mammalian Lass6 and its related family members regulate synthesis of specific ceramides
Authors: Mizutani Yukiko , Kihara Akio , Igarashi Yasuyuki
Published: August 2005

The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides. Lass5 and Lass6 produced shorter ceramide species (C14:0- and C16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.
Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis
Authors: Cheung Joanne C. , Li Jing , Reithmeier Reinhart A. F.
Published: August 2005

Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1–4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (ΔA400–A408) in TM1. These results suggest that the TM2–3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1–2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2–3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2–3 suggest that these segments form a re-entrant loop in human AE1.
Post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications
Authors: Christensen Brian , Nielsen Mette S. , Haselmann Kim F. , Petersen Torben E. , Sørensen Esben S.
Published: August 2005

OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser8, Ser10, Ser11, Ser46, Ser47, Thr50, Ser60, Ser62, Ser65, Ser83, Ser86, Ser89, Ser92, Ser104, Ser110, Ser113, Thr169, Ser179, Ser208, Ser218, Ser238, Ser247, Ser254, Ser259, Ser264, Ser275, Ser287, Ser292 and Ser294) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser101, Ser107, Ser175, Ser199, Ser212 and Ser251) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser203, is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr118, Thr122, Thr127, Thr131 and Thr136) and two potential sites for N-glycosylation (Asn63 and Asn90) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.
Aurora-A site specificity: a study with synthetic peptide substrates
Authors: Ferrari Stefano , Marin Oriano , Pagano Mario A. , Meggio Flavio , Hess Daniel , El-Shemerly Mahmoud , Krystyniak Agnieszka , Pinna Lorenzo A.
Published: August 2005

AurA (Aurora-A) is a ubiquitous protein kinase regulating entry into mitosis and shown to promote transformation upon overexpression. In order to gain information on the structural features determining its substrate specificity, we assayed human recombinant AurA on a variety of phosphoacceptor peptide substrates including a series of properly modified derivatives of the Kemptide (ALRRASLGAA). The data presented here show that AurA is a basophilic Ser/Thr protein kinase recognizing the consensus R/K/N-R-X-S/T-B, where B denotes any hydrophobic residue with the exception of Pro. We show that the presence of a Pro at position n+1 fully abrogates phosphorylation of the peptide substrate. Although the consensus for AurA is reminiscent of that of PKA (protein kinase A), it significantly differs from the latter for a much more stringent dependence on the hydrophobic residue at n+1 and for its tolerance of residues other than Arg at position n−3. Based on the finding that the peptide ALKRASLGAA is not a substrate of PKA while still providing a sensitive assay of AurA activity, we suggest that this peptide may be used for differential screening of the two kinases. We have further validated the AurA consensus by generating a peptide (APSSRRTT288LCGT) that comprises the main AurA autophosphorylation site and by showing that AurA phosphorylated this peptide exclusively at one site fulfilling its consensus (Thr288). Moreover, we show that AurA could autophosphorylate at Thr288 through an intermolecular mechanism of reaction and that, in vivo, PKA was not involved with Thr288 phosphorylation. The evidence obtained in the present study provides a rational tool for predicting AurA sites in potential substrates of physiological significance.
Metal ion modulation of cystinyl aminopeptidase
Authors: Laeremans Hilde , Demaegdt Heidi , De Backer Jean-Paul , Le Minh T. , Kersemans Veerle , Michotte Yvette , Vauquelin Georges , Vanderheyden Patrick M. L.
Published: August 2005

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2′-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32±1.78 and 2.10±0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N′,N″,N″-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as ‘AT4 receptor binding’, only occurs for the cystinyl aminopeptidase apoenzyme.