Search results for "Ethylmaleimide"
showing 10 items of 14 documents
In vivo effect of N-ethylmaleimide (NEM) on the measurement of nitrate in plasma
2016
Bioavailability of nitric oxide in the body may be estimated by measuring the concentration of nitrate in plasma. However, it has not been reported whether sequestering of aminothiols in plasma affects the concentration of nitrate in the samples.N-ethylmaleimide (NEM) sequesters aminothiols in plasma therefore we tested the in vivo effect of NEM on the concentration nitrate in plasma.Blood samples were collected from 56 healthy subjects in EDTA vials, EDTA vials containing PBS (pH7.4) and EDTA vials containing NEM dissolved in PBS. Nonparametric statistical tests were used to study the effect of NEM on the concentration of nitrate in plasma measured by the Griess reagent assay and by an HPL…
INFγ stimulates arginine transport through system y+L in human monocytes
2004
Freshly isolated human monocytes transport L-arginine mostly through a sodium independent, NEM insensitive pathway inhibited by L-leucine in the presence, but not in the absence of sodium. Interferon-gamma (IFNgamma) stimulates this pathway, identifiable with system y+L, and markedly enhances the expression of SLC7A7, the gene that encodes for system y+L subunit y+LAT1, but not of SLC7A6, that codes for the alternative subunit y+LAT2. System y+ plays a minor role in arginine uptake by monocytes and the expression of system y+-related genes, SLC7A1 and SLC7A2, is not changed by IFNgamma. These results demonstrate that system y+L is sensitive to IFNgamma.
Characterization of fusion from without induced by herpes simplex virus
1991
The process of fusion from without (FFWO) induced by herpes simplex virus (HSV) was analyzed by using various inhibitors and compared to fusion from within (FFWI). The fate of certain elements of the cytoskeleton after FFWO was also investigated. Our experiments demonstrate FFWO as a very suitable system for study of early virus-cell interactions. Zn++ ions proved inhibitory for penetration whilst pretreatment of cells with Ca++ ions before infection enhanced FFWO activity. Dissociation of penetration from the fusion process itself was possible by use of Zn++ ions, low pH-treatment and antiserum on the one hand and N-ethylmaleimide and cytochalasin D on the other. Penetration itself needs o…
Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of …
1996
Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5'-[gamma-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide(NEM)-sensitive fraction. The NEM-…
Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide
2013
In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y(+)L (y(+)LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y(+) (most likely CAT-1), but not system y(+)L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753-766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y(+)LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y(+)LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced tran…
Coupling of Contact Sensitizers to Thiol Groups is a Key Event for the Activation of Monocytes and Monocyte-Derived Dendritic Cells
2003
Strong contact sensitizers are able to induce distinct signal transduction mechanisms in antigen-presenting cells by coupling to cell proteins. The predominant target structures of haptens are thought to be thiol and amino groups in cysteine and lysine residues. We studied whether coupling of small reactive chemicals to thiol or amino groups might be responsible for the activation of monocytes and mature monocyte-derived dendritic cells. Human peripheral blood mononuclear cells were stimulated in vitro with subtoxic concentrations of the strong haptens 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone and 2, 4, 6-trinitrochlorobenzene, the thiol-reactive reagents N-hydroxymaleim…
Activation of L-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages
1998
1 The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. 2 L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β-Phorbol 12-myristate 13-acetate (PMA), but not 4α-phorbol 12-myristate 13-acetate (α-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. 3 L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. 4 Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This…
The Effects of Sulphydryl Reagents on the Binding and Mixed Function Oxidation of Hexobarbital in Rat Hepatic Microsomes
1975
1. The effects of the sulphydryl reagents p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide on the binding spectrum, oxygen consumption and formation of a suspected substrate-cytochrome P-450-oxygen complex for hexobarbital in rat liver microsomes were investigated. 2. The oxygen consumption caused by hexobarbital oxidation was inhibited non-competitively by all three agents, with 50% inhibition at 4 times 10(-5) M for p-chloromercuribenzoate, 3-7 times 10(-4) M for N-ethylmaleimide and 1-9 times 10(-3) M for iodoacetamide. Cysteamine protected and at least partially reversed this inhibition. 3. p-chloromercuribenzoate inhibited the formation of the cytochrome P-450-substrate-oxyg…
Sulfhydryl G Proteins and Phospholipase A2-Associated G Proteins Are Involved in Adrenergic Signal Transduction in the Rat Pineal Gland
2001
The rat pineal gland with its circadian noradrenaline-regulated melatonin rhythm is an excellent model for studying adrenergic signal transduction with respect to cAMP and cGMP formation. The stimulatory G(s) proteins play a well-established role in this process. In contrast, the potential roles of the inhibitory G(i) proteins, the functionally unclear other G(o) proteins, and a number of G protein subtypes are not known. The present study examines the effects on beta(1)- and beta(1)-plus-alpha(1)-stimulated cAMP and cGMP formation of a number of G protein modulators in rat pinealocyte suspension cultures. The effects of the nitric oxide donor sodium nitroprusside on cGMP were also examined…
Topology and accessibility of the transmembrane helices and the sensory site in the bifunctional transporter DcuB of Escherichia coli.
2011
C(4)-Dicarboxylate uptake transporter B (DcuB) of Escherichia coli is a bifunctional transporter that catalyzes fumarate/succinate antiport and serves as a cosensor of the sensor kinase DcuS. Sites and domains of DcuB were analyzed for their topology relative to the cytoplasmic or periplasmic side of the membrane and their accessibility to the water space. For the topology studies, DcuB was fused at 33 sites to the reporter enzymes PhoA and LacZ that are only active when located in the periplasm or the cytoplasm, respectively. The ratios of the PhoA and LacZ activities suggested the presence of 10 or 11 hydrophilic loops, and 11 or 12 α-helical transmembrane domains (TMDs). The central part…