bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma patch structuring by the nonlinear evolution of the gradient drift instability in the high‐latitude ionosphere
Authors: Gondarenko N. A. , Guzdar P. N.
Published: September 2004

Results from the three‐dimensional nonlinear simulations of the gradient drift instability to study structuring in high‐latitude plasma patches are presented. Simulations demonstrate that the existence of the mesoscale structures (10 km to 100 m) on both leading and trailing edges and inside a patch is due to (1) the nonlinear development of the gradient drift instability (GDI) or due to (2) occasional reversal of the direction of convection. The high‐resolution simulation data have a dynamic range that allows us to make detailed comparisons with the observed density and velocity fluctuation spectra. We use the measured ion density and horizontal velocity data from the Dynamic Explorer 2 (DE 2) spacecraft examined by Kivanç and Heelis [1997] to compare them with our simulations. Density structures in the leading and trailing edges of patches have been examined, and comparison of statistical characteristics of the average density gradient on these edges with those in the work by Coley and Heelis [1998] has been performed. The dynamics and evolution of the density fluctuations over the realistic time scale are shown, thereby providing a significant evidence of the existence of the GDI at high latitudes as a structure‐generating mechanism and a first principle understanding of the hierarchy of the instabilities involved in the observed structuring in high‐latitude plasma patches.
Estimates of atmospheric‐processed soluble iron from observations and a global mineral aerosol model: Biogeochemical implications
Authors: Hand J. L. , Mahowald N. M. , Chen Y. , Siefert R. L. , Luo C. , Subramaniam A. , Fung I.
Published: September 2004

Desert dust deposition to the ocean may be a significant source of biogeochemically important elements, specifically iron. The bioavailability of iron in the oceans requires it to be in a soluble form, and because atmospheric iron in desert dust is typically insoluble, understanding the atmospheric processes that convert insoluble iron to more soluble forms is essential. Understanding these relationships is especially important in remote ocean regions where iron may be the limiting nutrient. Observations of soluble iron from 2001 cruise‐based aerosol measurements over the Atlantic and Pacific Oceans ranged from 0 to 45% (mean of 4 ± 9%) in the fine mode (<2.5 μm in diameter) and 0 to 87% (mean of 2 ± 10%) in the coarse mode. We test two simple hypotheses of soluble iron enhancement in the atmosphere using a global model of mineral aerosols. The first method assumes that iron solubility increases as iron is exposed to solar radiation, approximating photoreduction reactions that are important pathways for enhancement of soluble iron in the presence of acidic solutions. The second process imitates cloud processing of iron by increasing the amount of soluble iron when the mineral aerosol comes into contact with a cloud. Both methods resulted in similar average magnitudes of percent soluble iron compared to observations but did not capture specific events or have sufficient variability, perhaps because the model does not include aerosol interactions between species other than mineral dust or other processes that may be important.
Comparison of two radiation algorithms for surface‐based cloud‐free sky detection
Authors: Sutter Marcel , Dürr Bruno , Philipona Rolf
Published: September 2004

Two methods to automatically detect cloud‐free skies from the Earth's surface, which are based on algorithms using surface radiation measurements, are compared with Weather Service ground observations and each other. One algorithm uses total and diffuse shortwave downward radiation (referred to as the shortwave radiation algorithm (SWRA)), and the other uses longwave downward radiation in combination with surface temperature and humidity (referred to as the longwave radiation algorithm (LWRA)). The data used for the comparison originate from five Baseline Surface Radiation Network (BSRN) stations, two in polar regions, one in central Europe, and two at low latitudes, covering time periods of 2–6 years. After determination of the parameterization both algorithms can be applied without further effort, offering a straightforward technique to identify cloud‐free skies over long time periods. With Weather Service ground observations as reference, LWRA detects cloud‐free skies around 80% of the time. Significant overestimation of cloud cover happens during strong inversions. Thin high clouds on the one hand and dry haze (in an otherwise cloud‐free sky) on the other hand can cause LWRA to wrongly calculate a clear sky. The percentage of detected and missed cloud‐free skies by SWRA depends on the parameterization (i.e., the definition of cloud‐free sky). Compared to Weather Service ground observations, we noticed lower rates of detected cloud‐free skies at stations with frequent events with increased turbidity and underestimation of cloud cover occasionally during periods with only thin high clouds, especially if they are accompanied by increased turbidity.
Measurements of organic and elemental carbon in Asian outflow during ACE‐Asia from the NSF/NCAR C‐130
Authors: Huebert Barry , Bertram Timothy , Kline Jena , Howell Steven , Eatough Delbert , Blomquist Byron
Published: September 2004

During the spring 2001 Asian Pacific Regional Aerosol Characterization Experiment (ACE‐Asia) intensive observation period, we measured elemental and organic carbon (EC and OC) from the National Science Foundation/National Center for Atmospheric Research (NSF/NCAR) C‐130 aircraft. Samples were collected on quartz filters using a Particle Concentrator–Brigham Young University Organic Sampling System (PC‐BOSS) to minimize positive and negative artifacts and analyzed using a Sunset Labs thermal/optical analyzer. Although short aircraft sampling times limited our sensitivity, total carbon (EC plus OC) was quantifiable on all but one level flight leg. We could not confidently apportion total carbon (TC) between EC and OC on about a quarter of the legs, mostly in the free troposphere (FT). About a third of the samples showed significant evaporation of collected aerosols from their quartz filter (a median of 20% on these samples), while a similar number had too little evaporation to detect. We found that TC and OC concentrations were generally higher in the boundary layer (medians of 7.6 μg TC/m3 and 5.8 μg OC/m3) than in the free troposphere (medians of 3.1 μg TC/m3 and 3.9 μg OC/m3, the latter from only a few samples). The same appears to be true for EC, but the free troposphere (FT) statistics for EC are also poor. Average concentrations were somewhat higher than the medians, reflecting the impact of a few more polluted samples. OC was much more variable than was EC. In the FT the TC to non‐sea‐salt sulfate ratio ranged from 3.2 to 6.0, so carbonaceous aerosols were considerably more concentrated than non‐sea‐salt sulfate in the upper troposphere. When dust mass was included, organic matter ranged from about 0.1 to 0.7 of the fine particle mass. TC/EC ratios ranged from 2 to 15, with medians of ∼4–5. Air in the Yellow Sea, the Korea Strait, and the Sea of Japan had generally higher concentrations of OC than that in the East China Sea or the Pacific south and east of Japan.
Florida thunderstorms: A faucet of reactive nitrogen to the upper troposphere
Authors: Ridley B. , Ott L. , Pickering K. , Emmons L. , Montzka D. , Weinheimer A. , Knapp D. , Grahek F. , Li L. , Heymsfield G. , McGill M. , Kucera P. , Mahoney M. J. , Baumgardner D. , Schultz M. , Brasseur G.
Published: September 2004

During the July 2002 Cirrus Regional Study of Tropical Anvils and Cirrus Layers‐Florida Area Cirrus Experiment (CRYSTAL‐FACE), flights of a WB‐57F aircraft revealed mixing ratios of nitric oxide 10–50 times background over distances of 25–175 km in the anvils of thunderstorms and in clear air downwind of storm systems due to lightning activity and possible transport from the boundary layer. Estimates of the total mass of NOx injected into the middle and upper troposphere differed considerably for a moderately versus highly electrically active storm system as expected. However, assuming that the total mass is dominated by lightning production, rough estimates of the production per average lightning flash for a moderately and a highly active storm also yielded quite different ranges of (0.33–0.66) × 1026 and (1.7–2.3) × 1026 molecules NO/flash, respectively. If the common assumption is made that intracloud flashes have 1/10th the NO production efficiency of cloud‐to‐ground (CG) flashes, the ranges of production for the moderately and highly active storms were (0.88–1.8) × 1026 and (4.5–6.1) × 1026 molecules NO/CG flash, respectively. The observed CG flash accumulations and NOx mass production estimate for the month of July 2002 over the Florida area are compared with results from the MOZART‐2 global chemistry‐transport model that uses a common lightning flash parameterization. Reasonable agreement was found after a correction to the lightning parameterization was made. Finally, broad‐scale median mixing ratios of NO within anvils over Florida were significantly larger than found in storms previously investigated over Colorado and New Mexico.
Structural double-mutant cycle analysis of a hydrogen bond network in ketosteroid isomerase from Pseudomonas putida biotype B
Authors: JANG Do Soo , CHA Hyung Jin , CHA Sun-Shin , HONG Bee Hak , HA Nam-Chul , LEE Ja Young , OH Byung-Ha , LEE Heung-Soo , CHOI Kwan Yong
Published: September 2004

KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp99···Water504···Tyr14···Tyr55···Tyr30, connects two critical catalytic residues, Tyr14 and Asp99, with Tyr30, Tyr55 and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The ΔΔGo values of Y14F/D99L (Tyr14→Phe/Asp99→Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr14 and Asp99 should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The ΔΔGo values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr30 and Asp99 or Tyr55 and Asp99 should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr14, involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.
Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca2+ release and entry in DT40 B lymphocytes
Authors: MORITA Takao , TANIMURA Akihiko , NEZU Akihiro , KUROSAKI Tomohiro , TOJYO Yosuke
Published: September 2004

We examined the function of GFP-IP3R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca2+ release and entry using a mutant DT40 cell line (IP3R-KO) in which all three IP3R genes had been disrupted. GFP-IP3R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP3R3 apparently co-localized with the plasma membrane. The application of IP3 to permeabilized WT (wild-type) DT40 cells induced Ca2+ release from internal stores. Although this did not occur in IP3R-KO cells it was restored by expression of GFP-IP3R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca2+ response in IP3R-KO cells, whereas these treatments induced oscillatory or transient Ca2+ responses in GFP-IP3R3-expressing IP3R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca2+ entry in WT and GFP-IP3R3-expressing IP3R-KO cells but not in IP3R-KO cells. This BCR-mediated Ca2+ entry was observed in the presence of La3+, which blocks capacitative Ca2+ entry. Thapsigargin depleted Ca2+ stores and led to Ca2+ entry in IP3R-KO cells irrespective of GFP-IP3R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca2+ entry was completely blocked by La3+, suggesting that the BCR-mediated Ca2+ entry pathway is distinct from the capacitative Ca2+ entry pathway. The present study demonstrates that GFP-IP3R3 could compensate for native IP3R in both IP3-induced Ca2+ release and BCR-mediated Ca2+ entry.
Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A
Authors: MAGIERA Maria M. , GUPTA Mona , RUNDELL Catherine J. , SATISH Nilima , ERNENS Isabelle , YARWOOD Stephen J.
Published: September 2004

Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (ΔcAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with ΔcAMP-EPAC1 and ΔDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.
Characterization and functional analysis of the 5′-flanking region of the mouse 20α-hydroxysteroid dehydrogenase gene
Authors: HIRABAYASHI Keiji , ISHIDA Maho , SUZUKI Masatoshi , YAMANOUCHI Keitaro , NISHIHARA Masugi
Published: September 2004

20α-Hydroxysteroid dehydrogenase (20α-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5′-flanking region of the mouse 20α-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5′-flanking region, revealed that the region between −83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wild-type Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20α-HSD gene.
Myristoylated alanine-rich C kinase substrate (MARCKS) is involved in myoblast fusion through its regulation by protein kinase Cα and calpain proteolytic cleavage
Authors: DULONG Sandrine , GOUDENEGE Sebastien , VUILLIER-DEVILLERS Karine , MANENTI Stéphane , POUSSARD Sylvie , COTTIN Patrick
Published: September 2004

MARCKS (myristoylated alanine-rich C kinase substrate) is a major cytoskeletal protein substrate of PKC (protein kinase C) whose cellular functions are still unclear. However numerous studies have implicated MARCKS in the stabilization of cytoskeletal structures during cell differentiation. The present study was performed to investigate the potential role of Ca2+-dependent proteinases (calpains) during myogenesis via proteolysis of MARCKS. It was first demonstrated that MARCKS is a calpain substrate in vitro. Then, the subcellular expression of MARCKS was examined during the myogenesis process. Under such conditions, there was a significant decrease in MARCKS expression associated with the appearance of a 55 kDa proteolytic fragment at the time of intense fusion. The addition of calpastatin peptide, a specific calpain inhibitor, induced a significant decrease in the appearance of this fragment. Interestingly, MARCKS proteolysis was dependent of its phosphorylation by the conventional PKCα. Finally, ectopic expression of MARCKS significantly decreased the myoblast fusion process, while reduced expression of the protein with antisense oligonucleotides increased the fusion. Altogether, these data demonstrate that MARCKS proteolysis is necessary for the fusion of myoblasts and that cleavage of the protein by calpains is involved in this regulation.
Human septin–septin interactions as a prerequisite for targeting septin complexes in the cytosol
Authors: MARTÍNEZ Constantino , SANJUAN Miguel A. , DENT Judith A. , KARLSSON Lars , WARE Jerry
Published: September 2004

Septins are a cytosolic GTP-binding protein family first characterized in yeast, but gaining increasing recognition as critical protagonists in higher eukaryotic cellular events. Mammalian septins have been associated with cytokinesis and exocytosis, along with contributing to the development of neurological disorders. Ten different septins, divided into four groups, have been identified in mammals, and individual septins are capable of interacting with each other to form macromolecular complexes. The present study characterizes the structural requirements for human septin–septin interactions using a yeast two-hybrid system. We focus on three septins that are highly expressed in platelets and neurons, SEPT4 [previously designated H5, CDCrel-2 (cell-division-control-related-2), PNUTL2], SEPT5 (CDCrel-1, PNUTL1) and SEPT8 (KIAA0202). Each of these three septins contains a characteristic domain structure consisting of unique N- and C-termini, and a central core domain conserved among the family of proteins. The yeast two-hybrid system yielded data consistent with a model where each of the three septins can interact with itself (homotypic assembly) or with one of the other septins (heterotypic assembly). For SEPT5 and SEPT8, the results illustrate a model whereby heterotypic septin assembly is dependent on the conserved central core domain and homotypic interactions require the N- and C-termini of each protein. We also characterized a model in which the proper cellular localization of SEPT5 and SEPT8 requires concomitant expression of both proteins. Co-transfection of SEPT5 and SEPT8 results in both proteins targeted to a vesicular-like location. Therefore the cellular repertoire of human septins has an impact on function by targeting septin macromolecular complexes to specific cellular locations.
Cytochrome c oxidase subunit IV as a marker of protein kinase Cε function in neonatal cardiac myocytes: implications for cytochrome c oxidase activity
Authors: OGBI Mourad , CHEW Catherine S. , POHL Jan , STUCHLIK Olga , OGBI Safia , JOHNSON John A.
Published: September 2004

We have previously demonstrated that low concentrations of phorbol esters stimulate the selective translocation of protein kinase C (PKC) α and ε from the cell soluble to the particulate fraction in NCMs (neonatal rat cardiac myocytes). We therefore determined if the in vitro phosphorylation of substrates in these fractions could be used as assays of PKCα or ε activation. Intact cell phorbol ester treatment caused a decline in the in vitro32P-incorporation into several proteins in the cell-soluble fraction. These declines occurred in the presence or absence of in vitro Ca2+ and probably reflected the exit of PKC isoenzymes from the soluble fraction. In contrast, an approx. 18 kDa protein incorporated 32P in particulate fractions isolated from 4β-PMA-treated cells in a Ca2+-independent manner. Proteomic and immunoprecipitation analyses indicated that the protein is subunit IV of the cytochrome c oxidase complex (COIV). In vitro phosphorylation of COIV was attenuated by PKC pseudosubstrate peptides. Introduction of an PKCε-selective translocation inhibitor [Johnson, Gray, Chen and Mochly-Rosen (1996) J. Biol. Chem. 271, 24962–24966] into NCMs before 4β-PMA treatments also attenuated the in vitro phosphorylation of COIV. In mitochondrial extracts from 4β-PMA-treated NCMs, the PKCε isoenzyme coimmunoprecipitated with COIV, and cytochrome c oxidase activity was enhanced 2-fold. The in vitro phosphorylation of COIV reflects a novel approach for monitoring PKCε function in NCMs. Furthermore, PKCε probably interacts with COIV in NCM mitochondria to enhance electron-transport chain complex IV activity.
DNA binding, but not interaction with Bmal1, is responsible for DEC1-mediated transcription regulation of the circadian gene mPer1
Authors: LI Yuxin , SONG Xiulong , MA Yuzhong , LIU Jirong , YANG Dongfang , YAN Bingfang
Published: September 2004

DEC1 (differentially expressed in chondrocytes 1) and DEC2 are E-box-binding transcription factors and exhibit a circadian expression pattern. Recently, both proteins were found to repress the Clock/Bmal1-activated E-box promoters (e.g. mPer1). Yeast two-hybrid assay detected interactions between Bmal1 and DECs. It was hypothesized that DEC-mediated repression on the mPer1 promoter is achieved by binding to E-box elements and interacting with Bmal1. In the present study, we report that E-box binding rather than Bmal1 interaction is responsible for the observed repression. In the absence of Clock/Bmal1, both DEC1 and DEC2 markedly repressed the mPer1 promoter reporter; however, DNA-binding mutants showed no repressive activity. Similarly, DEC1, but not its DNA-binding mutants, repressed the Clock/Bmal1-induced activation. In addition, DEC1R58P, a DNA-binding mutant with Bmal1 interactivity, repressed neither the mPer1 reporter directly nor the Clock/Bmal1-induced activation, providing direct evidence that DNA binding, rather than Bmal1 interactions, is responsible for the repression on the mPer1 promoter. Furthermore, disruption of the Sp1 site in the proximal promoter of mPer1 increased the repression of DEC1 proteins. Previous studies with mouse DEC2 showed that this factor interacts with Sp1. These findings suggest that DEC proteins regulate the expression of mPer1 through E-box binding and Sp1 interaction. Alterations on circadian systems are increasingly recognized as important risk factors for disease initiation and progression, and the expression of Dec genes is rapidly induced by environmental stimuli and is highly increased in tumour tissues. Therefore de-regulated expression of DEC genes probably alters normal circadian rhythms and contributes significantly to the pathogenesis of many diseases including cancer.
Basic residues play key roles in catalysis and NADP+-specificity in maize (Zea maysL.) photosynthetic NADP+-dependent malic enzyme
Authors: DETARSIO Enrique , ANDREO Carlos S. , DRINCOVICH María F.
Published: September 2004

C4-specific (photosynthetic) NADP+-dependent malic enzyme (NADP+-ME) has evolved from C3-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP+-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP+-ME K255I (Lys-255→Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP+-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play ‘back-up’ roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP+ versus NAD+) of maize NADP+-ME by interacting with the 2′-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP+ or NAD+, as well as by the reciprocal inhibition constants of the competitive inhibitors 2′-AMP and 5′-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436→Ile) with wild-type NADP+-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP+-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.
The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain
Authors: IMAI Shin-ichi , KAI Masahiro , YAMADA Keiko , KANOH Hideo , SAKANE Fumio
Published: September 2004

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKδ1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKδ1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKδ1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKδ1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKδ1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKδ1.
Incomplete glycosylation and defective intracellular targeting of mutant solute carrier family 11 member 1 (Slc11a1)
Authors: WHITE Jacqueline K. , STEWART Abigail , POPOFF Jean-Francois , WILSON Shona , BLACKWELL Jenefer M.
Published: September 2004

Solute carrier family 11 member 1 (Slc11a1, formerly Nramp1) is a highly glycosylated, 12 transmembrane domain protein expressed in macrophages. It resides in the membrane of late endosomes and lysosomes, where it functions as a bivalent cation transporter. Mice susceptible to infection by various intracellular pathogens including Leishmania donovani and Salmonella typhimurium carry a glycine to aspartic acid substitution at position 169 (G169D, Gly169→Asp), within transmembrane domain 4 of Slc11a1. To investigate the molecular pathogenesis of infectious disease susceptibility, we compared the behaviour of heterologously and endogenously expressed wild-type and mutant Slc11a1 by immunofluorescence, immunoelectron microscopy and Western-blot analysis. We found occasional late endosome/lysosome staining of mutant protein using immunoelectron microscopy, but most of the mutant Slc11a1 was retained within the ER (endoplasmic reticulum). Using glycosylation as a marker for protein maturation in two independent heterologous expression systems, we found that most mutant Slc11a1 existed as an ER-dependent, partially glycosylated intermediate species. Correct endosomal targeting of wild-type Slc11a1 continued despite disruption of N-glycosylation sites, indicating that glycosylation did not influence folding or sorting. We propose that the G169D mutation causes localized misfolding of Slc11a1, resulting in its retention in the ER and manifestation of the loss of function phenotype.
Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study
Authors: GUIGAS Bruno , DETAILLE Dominique , CHAUVIN Christiane , BATANDIER Cécile , De OLIVEIRA Frédéric , FONTAINE Eric , LEVERVE Xavier
Published: September 2004

Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition.
Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells
Authors: WU Chia-Mao , CHANG Hao-Teng , CHANG Margaret Dah-Tsyr
Published: September 2004

ECP (eosinophil cationic protein) is a major component of eosinophil granule proteins, and is used as a clinical biomarker for asthma and allergic inflammatory disease. ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known. In the present study, mECP–eGFP–6H, a recombinant fusion protein containing mature ECP (mECP), enhanced green fluorescence protein (eGFP) and a His6 tag (6H), has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP. We found that mECP–eGFP–6H entered into GH3 neuroendocrine cells and inhibited the growth of the cells with an IC50 of 0.8 μM. By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein–protein interaction between mECP and CPE (carboxypeptidase E), a well characterized metalloprotease. Further in vivo yeast two-hybrid screening has also revealed that residues 318–387 located in a region of unknown function in mature CPE are indispensable for association with mECP. In addition, the uptake of mECP–eGFP–6H is suppressed by dominant-negative expression of the recycling defect mutant pre-pro-HA–CPES471A,E472A in GH3 cells, suggesting that the entry of mECP–eGFP–6H is associated with the recycling of CPE in GH3 cells. Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cells, and such an endocytotic process allows the cytotoxic ECP to inhibit growth of the target cells.
Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases
Authors: GUY Mark R. , ILLARIONOV Petr A. , GURCHA Sudagar S. , DOVER Lynn G. , GIBSON Kevin J. C. , SMITH Paul W. , MINNIKIN David E. , BESRA Gurdyal S.
Published: September 2004

PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an α(1→6)LM intermediate through a PPM-dependent α(1→6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a ‘natural’ C75 polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial α(1→6)mannosyltransferase, to a synthetic Manp(1→6)-Manp-O-C10:1 disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis α(1→6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases.
Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1
Authors: FLEMING Ian N. , BATTY Ian H. , PRESCOTT Alan R. , GRAY Alex , KULAR Gursant S. , STEWART Hazel , DOWNES C. Peter
Published: September 2004

Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P2 to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P2 breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P3 concentrations, rather than the closely related lipid, PtdIns(3,4)P2. Finally, the data demonstrate that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.
Localization of Nox2 N-terminus using polyclonal antipeptide antibodies
Authors: PACLET Marie-Hélène , HENDERSON Lydia M. , CAMPION Yannick , MOREL Françoise , DAGHER Marie-Claire
Published: September 2004

Nox2/gp91phox (where phox is phagocyte oxidase) is the catalytic membrane subunit of the granulocyte NADPH oxidase complex involved in host defence. The current model of membrane topology of Nox2 is based upon the identification of glycosylation sites, of regions that interact with the regulatory cytosolic factors and of the epitopes recognized by antibodies. So far, the localization of the N-terminus of Nox2 was only speculative. In order to clarify this localization, we raised a polyclonal antiserum against the N-terminal sequence M1GNWVAVNEGL11. Purified antibodies recognize the mature protein as a broad band at 91 kDa (glycosylated form) or a band at 55 kDa after deglycosylation. Immunocytochemistry and flow-cytometry analysis show a strong binding of the anti-N-terminal antibodies to differentiated HL60 cells and neutrophils respectively, after permeabilization only. The N-terminus of Nox2 is therefore present in the mature protein and is located to the cytoplasmic side of the plasma membrane.
Extracellular NAD+ regulates intracellular free calcium concentration in human monocytes
Authors: GERTH Anja , NIEBER Karen , OPPENHEIMER Norman J. , HAUSCHILDT Sunna
Published: September 2004

Ca2+ ions play a critical role in the biochemical cascade of signal transduction pathways, leading to the activation of immune cells. In the present study, we show that the exposure of freshly isolated human monocytes to NAD+ results in a rapid concentration-dependent elevation of [Ca2+]i (intracellular free Ca2+ concentration) caused by the influx of extracellular Ca2+. NAD+ derivatives containing a modified adenine or nicotinamide ring failed to trigger a Ca2+ increase. Treating monocytes with ADPR (ADP-ribose), a major degradation product of NAD+, also resulted in a rise in [Ca2+]i. Selective inhibition of CD38, an NAD-glycohydrolase that generates free ADPR from NAD+, does not abolish the effect of NAD+, excluding the possibility that NAD+ might act via ADPR. The NAD+-induced Ca2+ response was prevented by the prior addition of ADPR and vice versa, indicating that both compounds share some mechanisms mediating the rise in [Ca2+]i. NAD+, as well as ADPR, were ineffective when applied following ATP, suggesting that ATP controls events that intersect with NAD+ and ADPR signalling.
Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site
Authors: VENÄLÄINEN Jarkko I. , JUVONEN Risto O. , GARCIA-HORSMAN J. Arturo , WALLÉN Erik A. A. , CHRISTIAANS Johannes A. M. , JARHO Elina M. , GYNTHER Jukka , MÄNNISTÖ Pekka T.
Published: September 2004

POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH2OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (Ki=11.8–0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, kon=(2.43±0.12)×105 M−1·s−1, whereas the inhibitor with a CN group exhibited the fastest binding, kon=(12.0±0.08)×105 M−1·s−1. In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH2OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the Ki values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.
Thrombangiitis obliterans: Eine Übersicht
Authors: Hoeft Daniela , Kroger Knut , Grabbe Stephan , Dissemond Joachim
Published: September 2004

Die Thrombangiitis obliterans (TAO) ist eine Erkrankung, die durch eine segmentale, nicht‐arteriosklerotische Entzündung der kleinen und mittleren Arterien und Venen charakterisiert ist. Die pathologischen vaskulären Veränderungen finden sich zumeist im Bereich der distalen Extremitäten, selten sind auch die Gefäße viszeraler Organe beteiligt. Betroffen sind vor allem junge männliche Raucher, bei denen es infolge einer Gefäßischämie zur Ausbildung von Ulzerationen und Gangrän der Zehen und Finger kommen kann. Die Diagnose wird aufgrund typischer klinischer Kriterien gestellt, sowie anhand von pathologischen Veränderungen, die sich angiographisch und histologisch nachweisen lassen. Die exakte Ätiologie der TAO ist bis heute unklar, es besteht jedoch eine nachgewiesene Assoziation mit Tabakkonsum. In der aktuellen Literatur wird auch eine autoimmunologische Genese diskutiert, wobei das initiierende Antigen bislang nicht identifiziert werden konnte.

Grundlage der Therapie der TAO ist der vollständige Verzicht auf jede Form des Tabakkonsums. Auf der Basis dieses Verzichtes kann eine medikamentöse Therapie, beispielsweise mit repetitiver Gabe von Prostacyclin‐Analoga eingeleitet werden. Die Lebenserwartung der Patienten mit TAO ist unverändert gegenüber der Normalbevölkerung, allerdings besteht bei einer Vielzahl der Patienten die Gefahr der Exulzeration mit nachfolgender Amputation.
15 % Azelainsäuregel in der Behandlung der Akne. Zwei doppelblinde klinische Vergleichsstudien
Authors: Gollnick Harald P. M. , Graupe Klaus , Zaumseil Rolf‐Peter
Published: September 2004

Hintergrund: Die topische Behandlung ist die Therapie erster Wahl für die milde bis mittelschwer ausgeprägte Acne vulgaris. Azelainsäure ist als 20%ige Cremeformulierung seit 15 Jahren als ein wirksames und sicheres Externum etabliert. Eine neue, nicht‐alkoholische Hydrogelformulierung mit 15 % Azelainsäure wurde klinisch gegenüber den Standardexterna 5 % Benzoylperoxid (BPO) und 1 % Clindamycin getestet.

Patienten und Methodik: In zwei unabhängigen, multizentrischen, randomisierten Vergleichsstudien bei 351 und 229 Patienten mit papulopustulöser Acne vulgaris wurde das 15 % Azleainsäuregel mit 5 % BPO‐ und 1 % Clindamycingel verglichen. Die Medikamente wurden zweimal täglich über 4 Monate angewendet.

Ergebnisse: Das Azelainsäuregel erwies sich dabei mit 70 bzw. 71 % prozentualer Reduktion der entzündlichen Effloreszenzen (Papeln und Pusteln) als genauso wirksam wie BPO und Clindamycin. Das Azelainsäuregel wurde gut vertragen; die Nebenwirkungen (lokales Brennen und Irritationszeichen) traten deutlich weniger als bei BPO, aber häufiger als bei Clindamycin auf. Trotz der Nebenwirkungen wurde das Azelainsäuregel von der überwiegenden Mehrheit der Patienten gut akzeptiert.

Schlußfolgerungen: Das Azelainsäuregel stellt eine wirksame topische Monotherapie der milden bis mittelschweren Acne vulgaris dar und ist in der neuen galenischen Formulierung eine Bereicherung in der Therapie der Akne.
Flow field topology of transient mixing driven by buoyancy
Authors: Duval Walter M. B.
Published: September 2004

Transient mixing driven by buoyancy occurs through the birth of a symmetric Rayleigh–Taylor morphology (RTM) structure for large length scales. Beyond its critical bifurcation the RTM structure exhibits self-similarity and occurs on smaller and smaller length scales. The dynamics of the RTM structure, its nonlinear growth and internal collision, show that its genesis occurs from an explosive bifurcation which leads to the overlap of resonance regions in phase space. This event shows the coexistence of regular and chaotic regions in phase space which is corroborated with the existence of horseshoe maps. A measure of local chaos given by the topological entropy indicates that as the system evolves there is growth of uncertainty. Breakdown of the dissipative RTM structure occurs during the transition from explosive to catastrophic bifurcation; this event gives rise to annihilation of the separatrices which drives overlap of resonance regions. The global bifurcation of explosive and catastrophic events in phase space for the large length scale of the RTM structure serves as a template for which mixing occurs on smaller and smaller length scales.
Refraction effects on the cavity formation and interaction of an intense ultra-short laser pulse with a gas jet
Authors: Hosokai Tomonao , Kinoshita Kenichi , Zhidkov Alexei , Nakamura Kei , Kotaki Hideyuki , Kando Masaki , Nakajima Kazuhisa , Uesaka Mitsuru
Published: September 2004

Formation of a plasma cavity with a shock wave in gas jets irradiated by tightly focused femtosecond laser pulses causes the wave break of the laser wake field at the front of the shock wave and, as a result, the injection of electrons into the acceleration phase of the wake-field wave. A strong crescentlike deformation of the cavity and a change in electron signal are observed with gas density growth. It is attributed to a mutual effect of the cavity on the laser pulse propagation and break of the plasma wake field due to refraction of the laser pulse.
Algorithmic information for interval maps with an indifferent fixed point and infinite invariant measure
Authors: Bonanno Claudio , Galatolo Stefano
Published: September 2004

Measuring the average information that is necessary to describe the behavior of a dynamical system leads to a generalization of the Kolmogorov–Sinai entropy. This is particularly interesting when the system has null entropy and the information increases less than linearly with respect to time. We consider a class of maps of the interval with an indifferent fixed point at the origin and an infinite natural invariant measure. We show that the average information that is necessary to describe the behavior of the orbits increases with time n approximately as nα, where α<1 depends only on the asymptotic behavior of the map near the origin.
Transport reduction by shear flows in dynamical models
Authors: Kim Eun-jin , Diamond P. H. , Hahm T. S.
Published: September 2004

The reduction in the transport of particles and heat by a strong mean shear flow is studied, in the context of interchange and ion-temperature gradient turbulence models. Compared to passive scalar transport, a stronger reduction in the transport (scaling with the shearing rate Ω as ∝Ω−3 ln Ω) results from a severe reduction in the amplitude of turbulent velocity in both models. However, the cross phase is only modestly reduced, as in the scalar field case. These results are in qualitative agreement with the results from both gyrokinetic and gyrofluid simulations of toroidal ion-temperature gradient turbulence [Lin et al., Phys. Rev. Lett. 83, 3645 (1999); Falchetto and Ottaviani, Phys. Rev. Lett. 92, 025002 (2004)], but contradict recent claims in some literature, highlighting the importance of the detailed properties of the flow in determining the overall transport level.