bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental investigation of acoustically enhanced solute transport in porous media
Authors: Vogler Eric T. , Chrysikopoulos Constantinos V.
Published: October 2002

The effect of acoustic waves on the transport of a conservative tracer in a water saturated column packed with glass beads was investigated. It was observed from the experimental data that the addition of acoustic waves, in the frequency range between 60 to 245 Hz, to a steady background pressure gradient, enhances solute transport compared to the base case consisting of only a background pressure gradient. Furthermore, it was found that the effective velocity of the solute is approximately inversely proportional to the frequency of the acoustic wave.
Glacier specific ablation rate derived by remote sensing measurements
Authors: Reeh Niels , Mohr Johan Jacob , Krabill William B. , Thomas Robert , Oerter Hans , Gundestrup Niels , Bøggild Carl Egede
Published: October 2002

The specific ablation rate in a transect of Storstrømmen, a large outlet glacier from the Northeast Greenland ice sheet, is derived by combining airborne laser altimetry measurements with emergence/submergence velocities derived from satellite synthetic aperture radar interferometry (InSAR) and airborne ice‐sounding radar measurement of ice thickness. The results are compared with in situ point observations of ablation measured at poles drilled into the glacier. Considering the different measurement periods for the different data sets, the agreement between derived and observed ablation rates is satisfactory. The new method of deriving specific mass balance, particularly ablation rate, by remote sensing measurements alone has the potential to substantially increase the density of observational based ablation rates on ice sheet and ice cap margins.
A generalized approach to parameterizing convection combining ensemble and data assimilation techniques
Authors: Grell Georg A. , Dévényi Dezső
Published: October 2002

A new convective parameterization is introduced that can make use of a large variety of assumptions previously introduced in earlier formulations. The assumptions are chosen so that they will generate a large spread in the solution. We then show two methods in which ensemble and data assimilation techniques may be used to find the best value to feed back to the larger scale model. First, we can use simple statistical methods to find the most probable solution. Second, the ensemble probability density function can be considered as an appropriate “prior” (a'priori density) for Bayesian data assimilation. Using this “prior”, and information about observation likelihood, measured meteorological or climatological data can be directly assimilated into model fields. Given proper observations, the application of this technique is not restricted to convective parameterizations, but may be applied to other parameterizations as well.
Winter Arctic Oscillation, Siberian High and East Asian Winter Monsoon
Authors: Wu Bingyi , Wang Jia
Published: October 2002

In this note, we investigate the impacts of the winter Arctic Oscillation (AO) and Siberian High (SH) on the East Asia winter monsoon (EAWM). It is found that the winter AO and the SH are relatively independent of each other in influencing the EAWM. The winter AO influences directly surface air temperature (SAT), sea level pressure (SLP) and the East Asian Trough at 500 hPa over the region northwards of 35°N in East Asia rather than through its impact on the SH. Compared with influences of the winter AO, the SH shows more direct and significant influences on the EAWM, particularly on SLP and northerly wind along the East Asian Coast. Impacts of the SH on the SAT occur primarily in the southwards of 50°N over East Asia, the northwestern Pacific and the South China Sea, because the AO suppresses the SH's influences in high latitudes of Asian Continent and the some subarctic regions.
Spatio‐temporal variation of the tidal triggering effect on earthquake occurrence associated with the 1982 South Tonga earthquake of Mw 7.5
Authors: Tanaka Sachiko , Ohtake Masakazu , Sato Haruo
Published: October 2002

We observed a clear tidal triggering effect on earthquakes closely related to the occurrence of the 1982 South Tonga earthquake (Mw 7.5) by precisely measuring the correlation between the earth tide and earthquake occurrence using 385 shallow earthquakes of reverse‐fault type in the Tonga‐Kermadec subduction zone. The result of statistical analysis indicates that the tidal effect, having been predominant for the normal stress on the fault plane, concentrated in and near the future focal area for several years preceding the South Tonga earthquake. The high correlation disappeared after the earthquake. The frequency distribution of phase angles for the pre‐event period exhibited a peak at the phase angle where the tidal normal stress is at its maximum to accelerate the fault slip, which indicates that the observed high correlation is not a statistical chance but physically reasonable.
Spatial patterns of variability in Antarctic surface temperature: Connections to the Southern Hemisphere Annular Mode and the Southern Oscillation
Authors: Kwok Ron , Comiso Josefino C.
Published: October 2002

The 17‐year (1982–1998) trend in surface temperature shows a general cooling over the Antarctic continent, warming of the sea ice zone, with moderate changes over the oceans. Warming of the peripheral seas is associated with negative trends in the regional sea ice extent. Effects of the Southern Hemisphere Annular Mode (SAM) and the extrapolar Southern Oscillation (SO) on surface temperature are quantified through regression analysis. Positive polarities of the SAM are associated with cold anomalies over most of Antarctica, with the most notable exception of the Antarctic Peninsula. Positive temperature anomalies and ice edge retreat in the Pacific sector are associated with El‐Niño episodes. Over the past two decades, the drift towards high polarity in the SAM and negative polarity in the SO indices couple to produce a spatial pattern with warmer temperatures in the Antarctic Peninsula and peripheral seas, and cooler temperatures over much of East Antarctica.
The present‐day deformation of the central Zagros from GPS measurements
Authors: Tatar Mohammad , Hatzfeld Denis , Martinod Joseph , Walpersdorf Andrea , Ghafori‐Ashtiany Mohsen , Chéry Jean
Published: October 2002

In 1997 and in 2000, we measured the distances between 14 geodetic benchmarks across the central Zagros mountain belt. The results show that about 10 mm/yr of shortening in the central Zagros is distributed across the mountain belt. This shortening corresponds to roughly 50% of the total convergence between Arabia and Eurasia and is consistent in direction. The Persian Gulf does not deform significantly. The Main Zagros Reverse Fault is not an active kinematic boundary. The internal deformation of the folded belt is rather homogeneous, at the scale of our survey, which does not allow us to detect any individual active blind fault. However, the strain pattern suggests that N‐S dextral strike slip faults may accommodate part of the deformation.
The influence of the Andes mountains on the South American low‐level flow
Authors: Campetella Claudia M. , Vera Carolina S.
Published: October 2002

A dry, hydrostatic, three‐dimensional primitive equation model is used to evaluate the mechanical effect of the Andes mountains on the South American low‐level flow (LLF). The model simulations reproduce the evolution of a baroclinic wave over the continent under winter and summer conditions. In both seasons as a consequence of the interaction between the basic flow and the Andes, northerly LLF was reproduced east of the Andes which resembles the main characteristics of the observed flow. Moreover, seasonal changes of cyclone activity have a significant impact on the location of maximum LLF and its alignment, which exhibits a NW‐SE orientation during winter and more meridional orientation in the warm season.
Contaminant‐induced mechanical damping in partially saturated Berea sandstone
Authors: Brunner W. M. , Spetzler H. A.
Published: October 2002

We have measured mechanical damping in partially saturated Berea sandstone that is strongly dependent on the presence of a small amount of oil. This effect is observed as a function of water saturation and average strain amplitude. These observations are presented as evidence of a damping mechanism previously observed and characterized in artificial cracks. We conclude that this damping effect is due to surface chemistry changes in the rock, and infer that seismic attenuation can be used to monitor small changes in pore fluid chemistry under certain conditions.
Analytical simulation of edge waves observed around the Balearic Islands
Authors: Liu P. L.‐F. , Monserrat S. , Marcos M.
Published: October 2002

We present sea level spectra computed from bottom pressure records at two locations around the Balearic Islands, Western Mediterranean. In these spectra, several sea level oscillation peaks are observed at both sides of Mallorca Island. Moreover, the relative magnitudes of the first two peaks switch during the “rissaga” events. To understand the appearance of these modes and their changes during ‘rissaga’, a simple analytical model is presented for atmospheric pressure induced edge waves on a circular shelf with a circular island in the middle. The analytical solutions indicate that indeed the peaks in the observed spectra can be identified as the fundamental modes of the edge waves. The analytical solutions also confirm that the magnitude of the second peak becomes higher than the first peak only when the phase speed of the atmospheric pressure waves become almost the same as that of the long ocean wave on the Mallorca shelf.
Cosmic rays and stratospheric aerosols: Evidence for a connection?
Authors: Vanhellemont Filip , Fussen Didier , Bingen Christine
Published: October 2002

In the search for the hypothetical missing link between a variety of atmospheric parameters and solar variability, we apply a Granger causality test to monthly averages of neutron counts (a proxy for cosmic rays), as measured at the Climax station, and stratospheric aerosol number densities, derived from extinction coefficients measured by the Stratospheric Aerosol and Gas Experiment II. A causal connection between the two time series is suggested. Further study of the cross‐correlation coefficient confirms this finding, indicating that cosmic rays influence stratospheric aerosols through a process with a time response of a few months. Our results have important implications for the hypothesis of a physical mechanism that links both quantities.
Increase of the tail plasma content during the northward interplanetary magnetic field intervals: Case studies
Authors: Nishino Masaki N. , Terasawa Toshio , Hoshino Masahiro
Published: October 2002

We have analyzed the structure of the magnetotail current sheet during the northward interplanetary magnetic field (IMF) intervals, using the method devised by Sergeev et al. [1998]. Case studies suggest that systematic variations of the current sheet thickness are seen not only during substorm activities but also during a building up phase of the cold dense plasma sheet when the IMF is northward and the magnetosphere is geomagnetically quiet. It is further found that during such northward IMF intervals the plasma “vertical content,” namely the product N0λ with the plasma density N0 at the plasma sheet center and the characteristic thickness λ, showed an order‐of‐magnitude increase within several hours. We estimate plasma transport rate during the northward IMF to be as much as 1026/s, which cannot be neglected in comparison with that during the southward IMF (∼1027 s−1).
Investigation of the role of the snowpack on atmospheric formaldehyde chemistry at Summit, Greenland
Authors: Dassau Terra M. , Sumner Ann Louise , Koeniger Stormy L. , Shepson Paul B. , Yang Jie , Honrath Richard E. , Cullen Nicolas J. , Steffen Konrad , Jacobi Hans‐Werner , Frey Markus , Bales Roger C.
Published: October 2002

Ambient gas‐phase and snow‐phase measurements of formaldehyde (HCHO) were conducted at Summit, Greenland, during several summers, in order to understand the role of air‐snow exchange on remote tropospheric HCHO and factors that determine snowpack HCHO. To investigate the impact of the known snowpack emission of HCHO, a gas‐phase model was developed that includes known chemistry relevant to Summit and that is constrained by data from the 1999 and 2000 field campaigns. This gas‐phase‐only model does not account for the high ambient levels of HCHO observed at Summit for several previous measurement campaigns, predicting approximately 150 ppt from predominantly CH4 chemistry, which is ∼25–50% of the observed concentrations for several years. Simulations were conducted that included a snowpack flux of HCHO based on HCHO flux measurements from 2000 and 1996. Using the fluxes obtained for 2000, the snowpack does not appear to be a substantial source of gas‐phase HCHO in summer. The 1996 flux estimates predict much higher HCHO concentrations, but with a strong diel cycle that does not match the observations. Thus, we conclude that, although the flux of HCHO from the surface likely has a significant impact on atmospheric HCHO above the snowpack, the time–dependent fluxes need to be better understood and quantified. It is also necessary to identify the HCHO precursors so we can better understand the nature and importance of snowpack photochemistry.
Modified Chebyshev planar arrays
Authors: Kabalan Karim Y. , El‐Hajj Ali , Al‐Husseini Mohammed
Published: October 2002

In this paper, the modified Chebyshev planar arrays are introduced. Like Chebyshev planar arrays, they have the important property of providing a constant sidelobe level in all directions. A formula for determining the current distribution in their elements is presented, and an exact formula for directivity is given for even and odd numbers of elements and any direction of maximum radiation. Compared to Chebyshev planar arrays with the same number of elements, same size, and same sidelobe level, the modified Chebyshev planar arrays provide a higher directivity when the number of elements exceeds a minimum value for the specified sidelobe level and interelement spacing. Numerical examples are given to illustrate the features of this new class of planar arrays and to compare their directivity and beam width with those of conventional Chebyshev planar arrays.
Solid precipitation on a tropical glacier in Bolivia measured with an ultrasonic depth gauge
Authors: Sicart Jean Emmanuel , Ribstein Pierre , Chazarin Jean Philippe , Berthier Etienne
Published: October 2002

An ultrasonic depth gauge was used to measure snowfall over a 2‐year period near the equilibrium line of the Zongo glacier (2.4 km2), Bolivia (16°S). Study of the influence of wind, air temperature, and air moisture on the measurements gives a quantification of snowfall at a 3‐hour time step, with a sensitivity of 1 cm of snow. The density of fresh snow is estimated by comparison with rain gauge measurements. The year is marked by a dry season from May to August and a wet season from December to April, during which accumulation and melting coincide on the glacier. Snowfall events are associated with a wind of moderate speed from the valley (less than 4 m s−1). Masses of moist air originate in the Amazon basin. The orographic effect produces precipitation at midday in the Andean valleys and in the afternoon in the high mountains. Nighttime snowfall events occur during periods of bad weather related to the regional atmospheric circulation and last several days. The density of fresh snow is high, about 250 kg m−3, because of the high air temperature during snowfall events (over −3°C). The high snow density and the moderate wind speeds prevent snow drifting conditions, which results in low spatial variability of the accumulation on tropical glaciers. Accurate recording of snowfall at a short time step is important for the study of energy fluxes at the glacier surface because snowfall events greatly increase the albedo and solar radiation is generally the main source of melting energy.
The heavy chain of neonatal Fc receptor for IgG is sequestered in endoplasmic reticulum by forming oligomers in the absence of β2-microglobulin association
Authors: ZHU Xiaoping , PENG Junmin , RAYCHOWDHURY Raktima , NAKAJIMA Atsushi , LENCER Wayne I. , BLUMBERG Richard S.
Published: October 2002

The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated with β2-microglobulin (β2m). In β2m-/- mice, FcRn functions are greatly impaired. We sought to determine how FcRn HC, particularly its structure and biogenesis, is affected by the absence of β2m. Human FcRn HC, expressed from the β2m-null cell line FO-1FcRn, was present as a monomeric 45-kDa protein under reducing conditions but primarily as a 92-kDa oligomeric protein under non-reducing conditions. Two-dimensional electrophoresis and MS analysis showed that the 92-kDa protein was a dimer of the 45-kDa HC. Immunostaining showed that FcRn HC in FO-1FcRn was co-localized with the endoplasmic reticulum (ER) protein Bip/GRP78 but not with an endosome protein, EEA1. In contrast, FcRn HC in FO-1FcRn+β2m was detected in both the ER and endosome. The dimeric HC in FcRn oligomers was free of β2m association in FO-1FcRn+β2m. Mutation of non-paired cysteine residues at positions 48 and 251 within the human FcRn cDNA failed to eliminate the oligomers. The FcRn HC oligomers could be reduced by reconstitution of FO-1FcRn with β2m or by balanced expression of FcRn HC with β2m, or β2m fused with a KDEL retention sequence. Similarly, the majority of FcRn HC isolated from neonatal β2m-/- mice was in a dimeric form under non-reducing conditions. The amount of FcRn HC was significantly decreased in β2m-/- mice and FO-1FcRn. Furthermore, β2m-free FcRn HC was sensitive to endoglycosidase digestion. These results indicate that FcRn HC alone can form disulphide-bonded oligomers in the ER, which may represent a misfolded protein. The β2m association with FcRn HC is critical for correct folding of FcRn and exiting the ER for routing to endosomes and the cell surface.
Trypanosoma brucei prenylated-protein carboxyl methyltransferase prefers farnesylated substrates
Authors: BUCKNER Frederick S. , KATEETE David P. , LUBEGA George W. , VOORHIS Wesley C. Van , YOKOYAMA Kohei
Published: October 2002

Carboxyl methylation of the C-terminal prenylated cysteine, which occurs in most farnesylated and geranylgeranylated proteins, is a reversible step and is implicated in the regulation of membrane binding and cellular functions of prenylated proteins such as GTPases. The gene coding for prenylated-protein carboxyl methyltransferase (PPMT) of the protozoan parasite Trypanosoma brucei has been cloned and expressed in the baculovirus/Sf9 cell system. The protein of 245 amino acids has 24—28% sequence identity to the orthologues from other species including human and Saccharomyces cerevisiae. Methyltransferase activity was detected in the membrane fraction from Sf9 cells infected with the recombinant baculovirus using N-acetyl-S-farnesylcysteine (AFC) and S-adenosyl[methyl-3H]methionine ([3H]AdoMet) as substrates. Recombinant T. brucei PPMT prefers AFC to N-acetyl-S-geranylgeranylcysteine (AGGC) by 10—50-fold based on the Vmax/Km values. Native PPMT activity detected in the membrane fraction from T. brucei procyclics displays similar substrate specificity (40-fold preference for AFC over AGGC). In contrast, mouse liver PPMT utilizes both AFC and AGGC as substrates with similar catalytic efficiencies. Several cellular proteins of the T. brucei bloodstream form were shown to be carboxyl methylated in a cell-free system. Incorporation of [3H]methyl group from [3H]AdoMet into most of the proteins was significantly inhibited by AFC but not AGGC at 20μM, suggesting that T. brucei PPMT acts on farnesylated proteins in the cell. Cells of the T. brucei bloodstream form show higher sensitivity to AFC and AGGC (EC50 = 70—80μM) compared with mouse 3T3 cells (EC50>150μM).
The role of thiol and nitrosothiol compounds in the nitric oxide-forming reactions of the iron–N-methyl-d-glucamine dithiocarbamate complex
Authors: TSUCHIYA Koichiro , KIRIMA Kazuyoshi , YOSHIZUMI Masanori , HOUCHI Hitoshi , TAMAKI Toshiaki , MASON Ronald P.
Published: October 2002

The object of the present study is to investigate whether the physiologically dominant thiol compounds such as GSH and cysteine or their nitrosothiol compounds affect the formation of the iron—N-methyl-d-glucamine dithiocarbamate [(MGD)2Fe2+]—nitric oxide complex. The present study provided experimental evidence that physiological concentrations of GSH (approx. 5mM) and l-cysteine (approx. 0.5mM) accelerated the formation of the (MGD)2Fe2+—NO complex from nitrite by two and three times respectively. The rate constants for the reduction of (MGD)3Fe3+ to (MGD)2Fe2+ by GSH and cysteine were calculated as 1.3 and 2.0×102M-1·s-1 respectively. Furthermore, depletion of GSH was demonstrated in PC12 cells, and thiol compounds enhanced the formation of reactive oxygen species by the (MGD)2Fe2+ complex by accelerating its redox turnover. The main effect of the physiological concentration of thiols was the reduction of (MGD)3Fe3+. S-nitrosoglutathione spontaneously reacted with (MGD)2Fe2+ to produce the (MGD)2Fe2+—NO complex with a 1:2 stoichiometry. In fact, (MGD)2Fe2+ was as good an indicator of nitrosothiols as it was of NO itself. The present study elucidates the difficulties of utilizing the (MGD)2Fe2+ complex for the quantification of NO in biological samples, especially in vivo.
Enhanced cAMP generation and insulin-releasing potency of two novel Tyr1-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes
Authors: GAULT Victor A. , FLATT Peter R. , BAILEY Clifford J. , HARRIOTT Patrick , GREER Brett , MOONEY Mark H. , O'HARTE Finbarr P.M.
Published: October 2002

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type II diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr1-modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P<0.01 to P<0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25nmol/kg) together with glucose (18mmol/kg) significantly reduced the peak 15min glucose excursion (1.4- and 1.5-fold respectively; P<0.05 to P<0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P<0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P<0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr1 modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type II diabetes mellitus.
JFC1 is transcriptionally activated by nuclear factor-κB and up-regulated by tumour necrosis factor α in prostate carcinoma cells
Authors: CATZ Sergio D. , BABIOR Bernard M. , JOHNSON Jennifer L.
Published: October 2002

The human promoter region of JFC1, a phosphatidylinositol 3,4,5-trisphosphate binding ATPase, was isolated by amplification of a 549bp region upstream of the jfc1 gene by the use of a double-PCR system. By primer extension analysis we mapped the transcription initiation site at nucleotide −321 relative to the translation start site. Putative regulatory elements were identified in the jfc1 TATA-less promoter, including three consensus sites for nuclear factor-κB (NF-κB). We analysed the three putative NF-κB binding sites by gel retardation and supershift assays. Each of the putative NF-κB sites interacted specifically with recombinant NF-κB p50, and the complexes co-migrated with those formed by the NF-κB consensus sequence and p50. An antibody to p50 generated a supershifted complex for these NF-κB sites. These sites formed specific complexes with nuclear proteins from tumour necrosis factor α (TNFα)-treated WEHI 231 cells, which were supershifted with antibodies against p50 and p65. The jfc1 promoter was transcriptionally active in various cell lines, as determined by luciferase reporter assays following transfection with a jfc1 promoter luciferase vector. Co-transfection with NF-κB expression vectors or stimulation with TNFα resulted in significant transactivation of the jfc1 promoter construct, although transactivation of a mutated jfc1 promoter was negligible. The expression of a dominant negative IκB (inhibitor κB) decreased basal jfc1 promoter activity. The cell lines PC-3, LNCaP and DU-145, but not Epstein—Barr virus-transformed lymphocytes, showed a dramatic increase in the expression of JFC1 after treatment with TNFα, suggesting that transcriptional activation of JFC1 by the TNFα/NF-κB pathway is significant in prostate carcinoma cell lines.
Intestinal mucins from cystic fibrosis mice show increased fucosylation due to an induced Fucα1-2 glycosyltransferase
Authors: THOMSSON Kristina A. , HINOJOSA-KURTZBERG Marina , AXELSSON Karin A. , DOMINO Steven E. , LOWE John B. , GENDLER Sandra J. , HANSSON Gunnar C.
Published: October 2002

In gene-targeted mouse models for cystic fibrosis (CF), the disease is mainly manifested by mucus obstruction in the intestine. To explore the mucus composition, mucins insoluble and soluble in 6M guanidinium chloride were purified by three rounds of isopycnic ultracentrifugation from the small and large intestines of CF mice (Cftrm1UNC/Cftrm1UNC) and compared with wild-type mice. The amino acid composition was typical of that for mucins and showed increased amounts of the insoluble (2.5-fold increase) and soluble (7-fold increase) mucins in the small intestine of the CF mice compared with wild-type mice. Mucins from the large intestine of both wild-type and CF mice showed a high but constant level of fucosylation. In contrast, the insoluble and soluble mucins of the small intestine in CF mice revealed a large increase in fucose, whereas those of wild-type mice contained only small amounts of fucose. This increased fucosylation was analysed by releasing the O-linked oligosaccharides followed by GC-MS. NMR spectroscopy revealed that the increased fucosylation was due to an increased expression of blood group H epitopes (Fucα1-2Gal-). Northern-blot analysis, using a probe for the murine Fucα1-2 fucosyltransferase (Fut2), showed an up-regulation of this mRNA in the small intestine of the CF mice, suggesting that this enzyme is responsible for the observed increase in blood group H-type glycosylation. The reason for this up-regulation could be a direct or indirect effect of a non-functional CF transmembrane conductance regulator (CFTR) caused by the absence of CFTR channel.
Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae
Authors: SEROV Alexander E. , POPOVA Anna S. , FEDORCHUK Vladimir V. , TISHKOV Vladimir I.
Published: October 2002

A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD+ to NADP+ has been constructed by introducing two single-point mutations, Asp196→Ala (D196A) and Tyr197→Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD+-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp196 and Tyr197 mediate the absolute coenzyme specificity of SceFDH for NAD+.
Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein
Authors: CORRADIN Sally , RANSIJN Adriana , CORRADIN Giampietro , BOUVIER Jacques , DELGADO Maria Belen , FERNANDEZ-CARNEADO Jimena , MOTTRAM Jeremy C. , VERGÈRES Guy , MAUËL Jacques
Published: October 2002

The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRPED) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRPED inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o-phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCKSED analogues containing modifications in the gp63 consensus cleavage site showed significant differences in inhibitory capacity. As phosphorylation of ED serine residues prevented gp63-mediated MRP degradation, we synthesized a pseudophosphorylated peptide in which serine residues were substituted by aspartate (3DMRPED). 3DMRPED was a highly effective inhibitor of both soluble and parasite-associated gp63. Finally, MRP ED peptides were synthesized together with an N-terminal HIV-1 Tat transduction domain (TD) to obtain cell-permeant peptide constructs. Such peptides retained gp63 inhibitory activity and efficiently entered both macrophages and parasites in a Tat TD-dependent manner. These studies may provide the basis for developing potent cell-permeant inhibitors of gp63.
Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes
Authors: LUIKEN J.J.F.P. , WILLEMS J. , COORT S.L.M. , COUMANS W.A. , BONEN A. , vanderVUSSE G.J. , GLATZ J.F.C.
Published: October 2002

Recently, we established that cellular contractions increase long-chain fatty-acid (FA) uptake by cardiac myocytes. This increase is dependent on the transport function of an 88kDa membrane FA transporter, FA translocase (FAT/CD36), and, in analogy to skeletal muscle, is likely to involve its translocation from an intracellular pool to the sarcolemma. In the present study, we investigated whether cAMP-dependent signalling is involved in this translocation process. Isoproterenol, dibutyryl-cAMP and the phosphodiesterase (PDE) inhibitor, amrinone, which markedly raised the intracellular cAMP level, did not affect cellular FA uptake, but influenced the fate of intracellular FAs by directing these to mitochondrial oxidation in electrostimulated cardiac myocytes. The PDE inhibitors 3-isobutyl-1-methylxanthine, milrinone and dipyridamole each significantly stimulated FA uptake as well as intracellular cAMP levels, but these effects were quantitatively unrelated. The stimulatory effects of these PDE inhibitors were antagonized by sulpho-N-succinimidylpalmitate, indicating the involvement of FAT/CD36, albeit that the different PDE inhibitors use different molecular mechanisms to stimulate FAT/CD36-mediated FA uptake. Notably, 3-isobutyl-1-methylxanthine and milrinone increased the intrinsic activity of FAT/CD36, possibly through its covalent modification, and dipyridamole induces translocation of FAT/CD36 to the sarcolemma. Elevation of intracellular cGMP, but not of cAMP, by the PDE inhibitor zaprinast did not have any effect on FA uptake and metabolism by cardiac myocytes. The stimulatory effects of PDE inhibitors on cardiac FA uptake should be considered when applying these agents in clinical medicine.
The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: identification of seven non-coding exons and characterization of a novel promoter
Authors: WHETSTINE Johnathan R. , FLATLEY Robin M. , MATHERLY Larry H.
Published: October 2002

Our previous study identified two alternate non-coding upstream exons (A and B) in the human reduced folate carrier (hRFC) gene, each controlled by a separate promoter. Each minimal promoter was regulated by unique cis-elements and transcription factors, including stimulating protein (Sp) 1 and Sp3 and the basic leucine zipper family of proteins, suggesting opportunities for cell- and tissue-specific regulation. Studies were performed to explore the expression patterns of hRFC in human tissues and cell lines. Levels of hRFC transcripts were measured on a multi-tissue mRNA array from 76 human tissues and tumour cell lines and on a multi-tissue Northern blot of representative tissues, each probed with full-length hRFC cDNA. hRFC transcripts were ubiquitously expressed, with the highest level in placenta and the lowest level in skeletal muscle. By rapid amplification of cDNA 5′-ends assay from nine tissues and two cell lines, hRFC transcripts containing both A and B 5′-untranslated regions (UTRs) were identified. However, five additional 5′-UTRs (designated A1, A2, C, D and E) were detected, mapping over 35kb upstream from the hRFC translation start site. The 5′-UTRs were characterized by multiple transcription start sites and/or alternative splice forms. At least 18 unique hRFC transcripts were detected. A novel promoter was localized to a 453bp fragment, including 442 upstream of exon C and 11bp of exon C. A 346bp repressor flanked the 3′-end of this promoter. Our results suggest an intricate regulation of hRFC gene expression involving multiple promoters and non-coding exons. Moreover, they provide a transcriptional framework for understanding the role of hRFC in the pathophysiology of folate deficiency and antifolate drug selectivity.
Purification, characterization and catalytic properties of human sterol 8-isomerase
Authors: NES W. David , ZHOU Wenxu , DENNIS Allen L. , LI Haoxia , JIA Zhonghua , KEITH Richard A. , PISER Timothy M. , FURLONG Stephen T.
Published: October 2002

CHO2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a Km of 50μM and a turnover number of 0.423s-1 for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Δ7-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using 2H2O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9α was established by a combination of 1H- and 13C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium (Keq = 14; ΔGo′ = −6.5kJ/mol) for double-bond isomerization favoured the forward direction, Δ8 to Δ7. Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants (Ki): emopamil (2μM) = tamoxifen (1μM) = tridemorph (1μM)<25-azacholesterol (21μM) <ketoconazole (156μM)<cholesterol (620μM). The results were consistent with stereoelectronic features of isomerization and support the general model for Δ7-sterol formation in cholesterol synthesis.
A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria
Authors: WILTSHIRE Carolyn , MATSUSHITA Masato , TSUKADA Satoshi , GILLESPIE David A.F. , MAY Gerhard H.W.
Published: October 2002

We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine—proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.
Characterization of an amyloid precursor protein-binding protein Fe65L2 and its novel isoforms lacking phosphotyrosine-interaction domains
Authors: TANAHASHI Hiroshi , TABIRA Takeshi
Published: October 2002

Human Fe65L2 is a member of the Fe65 protein family, which interacts with amyloid precursor protein (APP). Fe65L2 contains an N-terminal WW (Trp-Trp) domain followed by two phosphotyrosine-interaction domains, and consists of 486 amino acids. In the present study, we cloned and characterized two novel isoforms of Fe65L2, designated I-214 and I-245, which are produced by alternative splicing of the RNA. The splicing events disrupt the ability to bind with APP and low-density-lipoprotein-receptor-related protein. Fe65L2 was highly expressed in the brain, whereas I-214 and I-245 were expressed in various tissues. In HEK-293 cells, Fe65L2 was expressed in the nucleus and cytosol, whereas I-245 and I-214 were localized exclusively to the nucleus. The ratio of I-214 to Fe65L2 mRNA was increased by apoptotic stimuli. Although the overexpression of either Fe65L2 or I-214 did not significantly affect the half-life and maturation of APP, or the secretion of secreted APP, the secretion of β-amyloid peptide (Aβ)40 and Aβ42 was increased by overexpression of Fe65L2, but not by that of I-214. These results suggest that Fe65L2 affects Aβ production and a possible regulation of Fe65L2 function by alternative splicing.
Clostridium perfringens iota toxin: characterization of the cell-associated iota b complex
Authors: STILES Bradley G. , HALE Martha L. , MARVAUD Jean Christophe , POPOFF Michel R.
Published: October 2002

Clostridium perfringens type E iota toxin consists of two unlinked proteins designated as iota a (Ia; molecular mass47kDa), an ADP-ribosyltransferase and iota b (Ib; molecular mass81kDa) which binds to the cell surface and facilitates Ia entry into the cytosol. By Western-blot analysis, Ib incubated with Vero cells at 37°C generated a cell-associated, SDS-insoluble oligomer of Ib (molecular mass>220kDa) within 15s, which was still evident 110min after washing cells. Ib oligomerization was temperature, but not pH, dependent and was facilitated by a cell-surface protein(s). Within 5min at 37°C, cell-bound Ib generated Na+/K+ permeable channels that were blocked by Ia. However, Ib-induced channels or oligomers were not formed at 4°C. Two monoclonal antibodies raised against Ib that recognize unique, neutralizing epitopes within residues 632—655 either inhibited Ib binding to cells and/or oligomerization, unlike a non-neutralizing monoclonal antibody that binds within Ib residues 28—66. The Ib protoxin (molecular mass98kDa), which does not facilitate iota cytotoxicity but binds to Vero cells, did not oligomerize or form ion-permeable channels on cells, and neither trypsin nor chymotrypsin treatment of cell-bound Ib protoxin induced large complex formation. The link between Ib oligomers and iota toxicity was also apparent with a resistant cell line (MRC-5), which bound to Ib with no evidence of oligomerization. Overall, these studies revealed that the biological activity of iota toxin is dependent on a long-lived, cell-associated Ib complex that rapidly forms ion-permeable channels in cell membranes. These results further reveal the similarities of C. perfringens iota toxin with other bacterial binary toxins produced by Bacillus anthracis and C. botulinum.
Involvement of phospholipases D1 and D2 in sphingosine 1-phosphate-induced ERK (extracellular-signal-regulated kinase) activation and interleukin-8 secretion in human bronchial epithelial cells
Authors: WANG Lixin , CUMMINGS Rhett , USATYUK Peter , MORRIS Andrew , IRANI Kaikobad , NATARAJAN Viswanathan
Published: October 2002

Sphingosine 1-phosphate (S1P), a metabolite of sphingomyelin degradation, stimulates interleukin-8 (IL-8) secretion in human bronchial epithelial (Beas-2B) cells. The molecular mechanisms regulating S1P-mediated IL-8 secretion are yet to be completely defined. Here we provide evidence that activation of phospholipases D1 and D2 (PLD1 and PLD2) by S1P regulates the phosphorylation of extracellular-signal-regulated kinase (ERK) and IL-8 secretion in Beas-2B cells. S1P, in a time- and dose-dependent manner, enhanced the threonine/tyrosine phosphorylation of ERK. The inhibition of S1P-induced ERK phosphorylation by pertussis toxin and PD 98059 indicated coupling of S1P receptors to Gi and the ERK signalling cascade respectively. Treatment of Beas-2B cells with butan-1-ol, but not butan-3-ol, abrogated the S1P-induced phosphorylation of Raf-1 and ERK, suggesting that PLD is involved in this activation. The roles of PLD1 and PLD2 in ERK activation and IL-8 secretion activated by S1P were investigated by infecting cells with adenoviral constructs of wild-type and catalytically inactive mutants of PLD1 and PLD2. Infection of Beas-2B cells with the wild-type constructs resulted in the activation of PLD1 and PLD2 by S1P and PMA. Also, the enhanced production of [32P]phosphatidic acid and [32P]phosphatidylbutanol in the presence of butan-1-ol and the increased phosphorylation of ERK by S1P were blocked by the catalytically inactive mutants hPLD1-K898R and mPLD2-K758R. Transient transfection of Beas-2B cells with human PLD1 and mouse PLD2 cDNAs potentiated S1P-mediated IL-8 secretion compared with vector controls. In addition, PD 98059 attenuated IL-8 secretion induced by S1P in a dose-dependent fashion. These results demonstrate that both PLD1 and PLD2 participate in S1P stimulation of ERK phosphorylation and IL-8 secretion in bronchial epithelial cells.
Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)
Authors: WU Albert M. , WU June H. , TSAI Ming-Sung , LIU Jia-Hau , ANDRÉ Sabine , WASANO Kojiro , KALTNER Herbert , GABIUS Hans-Joachim
Published: October 2002

Galectins, a family of β-galactoside-specific endogenous lectins, are involved in regulating diverse activities such as proliferation/apoptosis, cell—cell (matrix) interaction and cell migration. It is presently unclear to what extent the carbohydrate fine specificities of the combining sites of mammalian galectins overlap. To address this issue, we performed an analysis of the carbohydrate-recognition domain (CRD-I) near the N-terminus of recombinant rat galectin-4 (G4-N) by the biotin/avidin-mediated microtitre plate lectin-binding assay with natural glycoproteins (gps)/polysaccharide and by the inhibition of galectin—glycan interactions with a panel of glycosubstances. Among the 35 glycans tested for lectin binding, G4-N reacted best with human blood group ABH precursor gps, and asialo porcine salivary gps, which contain high densities of the blood group Ii determinants Galβ1-3GalNAc (the mucin-type sugar sequence on the human erythrocyte membrane) and/or GalNAcα1-Ser/Thr (Tn), whereas this lectin domain reacted weakly or not at all with most sialylated gps. Among the oligosaccharides tested by the inhibition assay, Galβ1-3GlcNAcβ1-3Galβ1-4Glc was the best. It was 666.7 and 33.3 times more potent than Gal and Galβ1-3GlcNAc, respectively. G4-N has a preference for the β-anomer of Gal at the non-reducing ends of oligosaccharides with a Galβ1-3 linkage, over Galβ1-4 and Galβ1-6. The fraction of Tn glycopeptide from asialo ovine submandibular glycoprotein was 8.3 times more active than Galβ1-3GlcNAc. The overall carbohydrate specificity of G4-N can be defined as Galβ1-3GlcNAcβ1-3Galβ1-4Glc (lacto-N-tetraose)>Galβ1-4GlcNAcβ1-3Galβ1-4Glc (lacto-N-neo-tetraose) and Tn clusters>Galβ1-4Glc and GalNAcβ1-3Gal>Galβ1-3GalNAc>Galβ1-3GlcNAc>Galβ1-4GlcNAc>GalNAc>Gal. The definition of this binding profile provides the basis to detect differential binding properties relative to the other galectins with ensuing implications for functional analysis.
Pre- and post-translational negative effect of β-adrenoceptor agonists on adiponectin secretion: in vitro and in vivo studies
Authors: DELPORTE Marie-Laure , FUNAHASHI Tohru , TAKAHASHI Masahiko , MATSUZAWA Yuji , BRICHARD Sonia M.
Published: October 2002

The adipose-derived hormone, adiponectin (ApN), has a role in fuel homoeostasis, insulin action and atherosclerosis. Regulation of ApN by catecholamines has scarcely been investigated. We examined the effects of β-adrenergic agonists (and their second messenger, cAMP) on ApN gene expression, production and secretion in mouse in vitro and in vivo; their effects in human fat were also briefly studied in vitro. β-Adrenergic agonists and cAMP inhibited ApN gene expression in human visceral adipose tissue. Likewise, cAMP down-regulated ApN mRNAs in cultured mouse explants from visceral and subcutaneous regions. The amount of ApN released into the medium decreased concomitantly. cAMP also caused qualitative changes in ApN secretion. Under basal conditions, ApN was secreted as a single 32kDa species. In the presence of cAMP, an additional and probably immature (not modified post-translationally) 30kDa species was also sorted. This altered secretion resulted from cAMP-induced quantitative and qualitative changes of ApN within the adipocyte. Under basal conditions, the 32kDa form of ApN was mainly associated with high-density microsomes (HDMs), while the 30kDa species was confined to a pool recovered with the cytosol fraction. cAMP depleted intracellular ApN at the expense of both HDM and cytosol fractions, and abnormally targeted ApN species to the different subcellular compartments as a result of impaired maturation. β-Adrenergic agonists mimicked the inhibitory effects of cAMP on ApN mRNA and secretion, the β3-agonist BRL37344 being the most potent. Administration of BRL37344 to mice reduced ApN mRNAs in both adipose regions, and ApN levels in plasma. In conclusion, β-agonists inhibited ApN production and maturation, and thus exerted a dual (pre- and post-translational) negative effect on ApN secretion by cultured mouse adipose explants. ApN inhibition by β-agonists was reproduced in mouse in vivo and in humans in vitro. ApN down-regulation may have an important role in fuel homoeostasis, insulin resistance and stress-induced atherosclerosis.