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21.
Naotaka Sekiyama Andras Boeszoermenyi Haribabu Arthanari Gerhard Wagner Mélissa Léger-Abraham 《Biomolecular NMR assignments》2017,11(2):187-191
The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)4LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)4LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP144–87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m7GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction. 相似文献
22.
Manfred Keller Andras Seregi Georg Hertting Rolf Jackisch 《Neurochemistry international》1987,10(4):433-443
Prostaglandin (PG) and thromboxane B2 (TXB2) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD2 > TXB2 > PGF2 > PGE2, as measured by specific radioimmunoassays. Under basal conditions PGD2 biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A2 (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB2 biosynthesis depended on the availability of Ca2+, as demonstrated in Ca2+ free incubation medium containing Na2EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [3H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [3H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [3H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A2 and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.
In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca2+-dependent activation of phospholipase A2, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established. 相似文献
23.
24.
Lajos Attila Papp Mohammadhassan Foroughbakhshfasaei Béla Fiser Péter Horváth Eszter Kiss Khaled Sekkoum Árpád Gyéresi Gabriel Hancu Béla Noszál Zoltán-István Szabó Gergő Tóth 《Chirality》2020,32(2):158-167
A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers. 相似文献
25.
MOTIVATION: Prediction of disulfide bond connectivity facilitates structural and functional annotation of proteins. Previous studies suggest that cysteines of a disulfide bond mutate in a correlated manner. RESULTS: We developed a method that analyzes correlated mutation patterns in multiple sequence alignments in order to predict disulfide bond connectivity. Proteins with known experimental structures and varying numbers of disulfide bonds, and that spanned various evolutionary distances, were aligned. We observed frequent variation of disulfide bond connectivity within members of the same protein families, and it was also observed that in 99% of the cases, cysteine pairs forming non-conserved disulfide bonds mutated in concert. Our data support the notion that substitution of a cysteine in a disulfide bond prompts the substitution of its cysteine partner and that oxidized cysteines appear in pairs. The method we developed predicts disulfide bond connectivity patterns with accuracies of 73, 69 and 61% for proteins with two, three and four disulfide bonds, respectively. 相似文献
26.
Dosztányi Z Magyar C Tusnády GE Cserzo M Fiser A Simon I 《Nucleic acids research》2003,31(13):3359-3363
We describe several algorithms and public servers that were developed to analyze and predict various features of protein structures. These servers provide information about the covalent state of cysteine (CYSREDOX), as well as about residues involved in non-covalent cross links that play an important role in the structural stability of proteins (SCIDE and SCPRED). We also discuss methods and servers developed to identify helical transmembrane proteins from large databases and rough genomic data, including two of the most popular transmembrane prediction methods, DAS and HMMTOP. Several biologically interesting applications of these servers are also presented. The servers are available through http://www.enzim.hu/servers.html. 相似文献
27.
T cell activation-induced mitochondrial hyperpolarization is mediated by Ca2+- and redox-dependent production of nitric oxide 总被引:5,自引:0,他引:5
Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Deltapsi(m)) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Deltapsi(m) or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca(2+) levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca(2+) release, ROI levels, and NO production were diminished by inositol 1,4,5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (-85.0 +/- 10.0%; p = 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine elicited NO and ROI production, Ca(2+) release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5 +/- 0.8-fold; p = 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H(2)O(2) elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with beta-actin. H(2)O(2) also led to moderate mitochondrial hyperpolarization; however, Ca(2+) influx by ionomycin or Ca(2+) release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Deltapsi(m) elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI- and Ca(2+)-dependent NO production. 相似文献
28.
Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions 总被引:9,自引:0,他引:9
Bodo A Bakos E Szeri F Varadi A Sarkadi B 《The Journal of biological chemistry》2003,278(26):23529-23537
The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver. The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream. Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules. In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles. We found that the primary active transport of estradiol-17-beta-d-glucuronide (E217betaG), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3. Active E217betaG transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids. In contrast, all of these agents inhibited E217betaG transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217betaG, and the results indicated an allosteric cross-stimulation, probably a co-transport of bile acids and glucuronate conjugates through this protein. There was no such stimulation of bile acid transport by MRP3. In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver. 相似文献
29.
McKallip RJ Fisher M Do Y Szakal AK Gunthert U Nagarkatti PS Nagarkatti M 《The Journal of biological chemistry》2003,278(44):43818-43830
In the current study, we investigated the nature and role of CD44 variant isoforms involved in endothelial cell (EC) injury and tumor cell cytotoxicity mediated by IL-2-activated killer (LAK) cells. Treatment of CD44 wild-type lymphocytes with IL-2 led to increased gene expression of CD44 v6 and v7 variant isoforms and to significant induction of vascular leak syndrome (VLS). CD44v6-v7 knockout (KO) and CD44v7 KO mice showed markedly reduced levels of IL-2-induced VLS. The decreased VLS in CD44v6-v7 KO and CD44v7 KO mice did not result from differential activation and expansion of CD8+ T cells, NK, and NK-T cells or from altered degree of perivascular lymphocytic infiltration in the lungs. LAK cells from CD44v7 KO mice showed a significant decrease in their ability to adhere to and mediate lysis of EC but not lysis of P815 tumor cells in vitro. CD44v7-mediated lysis of EC by LAK cells was dependent on the activity of phosphatidylinositol 3-kinase and tyrosine kinases. Interestingly, IL-2-activated LAK cells expressing CD44hi but not CD44lo were responsible for EC lysis. Furthermore, lysis of EC targets could be blocked by addition of soluble or enzymatic cleavage of CD44v6-v7-binding glycosaminoglycans. Finally, anti-CD44v7 mAbs caused a significant reduction in the adherence to and killing of EC and led to suppression of IL-2-induced VLS. Together, this study suggests that the expression of CD44v7 on LAK cells plays a specific role in EC injury and that it may be possible to reduce EC injury but not tumor cell killing by specifically targeting CD44v7. 相似文献
30.
Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fcgamma receptors 总被引:4,自引:0,他引:4
Smith-Franklin BA Keele BF Tew JG Gartner S Szakal AK Estes JD Thacker TC Burton GF 《Journal of immunology (Baltimore, Md. : 1950)》2002,168(5):2408-2414
Large quantities of HIV are found trapped on the surface of follicular dendritic cells (FDCs), and virus persists on these cells until they ultimately die. We recently found that FDCs maintain HIV infectivity for long periods in vivo and in vitro. Because FDCs trap Ags (and virus) in the form of immune complexes and are rich in FcgammaRs, we reasoned that Ab and FcgammaRs may be required for FDC-mediated maintenance of HIV infectivity. To investigate this hypothesis, HIV immune complexes were formed in vitro and incubated for increasing times with or without FDCs, after which the remaining infectious virus was determined by HIV-p24 production in rescue cultures. FDCs maintained HIV infectivity in vitro in a dose-dependent manner but required the presence of specific Ab for this activity regardless of whether laboratory-adapted or primary X4 and R5 isolates were tested. In addition, Abs against either virally or host-encoded proteins on the virion permitted FDC-mediated maintenance of HIV infectivity. We found that the addition of FDCs to HIV immune complexes at the onset of culture gave optimal maintenance of infectivity. Moreover, blocking FDC-FcgammaRs or killing the FDCs dramatically reduced their ability to preserve virus infectivity. Finally, FDCs appeared to decrease the spontaneous release of HIV-1 gp120, suggesting that FDC-virus interactions stabilize the virus particle, thus contributing to the maintenance of infectivity. Therefore, optimal maintenance of HIV infectivity requires both Ab against particle-associated determinants and FDC-FcgammaRs. 相似文献