A conserved glutamate is important for slow inactivation in K+ channels.
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Voltage-gated ion channels undergo slow inactivation during prolonged depolarizations. We investigated the role of a conserved glutamate at the extracellular end of segment 5 (S5) in slow inactivation by mutating it to a cysteine (E418C in Shaker). We could lock the channel in two different conformations by disulfide-linking 418C to two different cysteines, introduced in the Pore-S6 (P-S6) loop. Our results suggest that E418 is normally stabilizing the open conformation of the slow inactivation gate by forming hydrogen bonds with the P-S6 loop. Breaking these bonds allows the P-S6 loop to rotate, which closes the slow inactivation gate. Our results also suggest a mechanism of how the movement of the voltage sensor can induce slow inactivation by destabilizing these bonds. (+info)
Transient growth inhibition of Escherichia coli K-12 by ion chelators: "in vivo" inhibition of ribonucleic acid synthesis.
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The ion chelators picolinic acid, quinaldic acid, 1,10-phenanthroline, and 8-hydroxyquinoline, but not ethylenediaminetetraacetate, ethyleneglycol-bis-(beta-aminoethyl ether)-N,N-tetraacetate, or dipicolinic acid, rapidly but transiently arrest growth of Escherichia coli K-12. Cells adapt and become resistant to growth inhibition by these agents, a process which requires protein synthesis. Mn2+, at low concentrations, decreases the time required for resumption of growth. Proteins synthesized during the lag are quantitatively and qualitatively different from those synthesized during normal growth. Inhibition of growth can explained by an effect on RNA polymerase, a known metalloenzyme. (+info)
Eotaxin is specifically cleaved by hookworm metalloproteases preventing its action in vitro and in vivo.
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Eotaxin is a potent eosinophil chemoattractant that acts selectively through CCR3, which is expressed on eosinophils, basophils, mast cells, and Th2-type T cells. This arm of the immune system is believed to have evolved to control helminthic parasites. We hypothesized that helminths may employ mechanisms to inhibit eosinophil recruitment, to prolong worm survival in the host. We observed that the excretory/secretory products of the hookworm Necator americanus inhibited eosinophil recruitment in vivo in response to eotaxin, but not leukotriene B(4), a phenomenon that could be prevented by the addition of protease inhibitors. Using Western blotting, N. americanus supernatant was shown to cause rapid proteolysis of eotaxin, but not IL-8 or eotaxin-2. N. americanus homogenate was fractionated by gel filtration chromatography, and a FACS-based bioassay measured the ability of each fraction to inhibit the activity of a variety of chemokines. This resulted in two peaks of eotaxin-degrading activity, corresponding to approximately 15 and 50 kDa molecular mass. This activity was specific for eotaxin, as responses to other agonists tested were unaffected. Proteolysis of eotaxin was prevented by EDTA and phenanthroline, indicating that metalloprotease activity was involved. Production of enzymes inactivating eotaxin may be a strategy employed by helminths to prevent recruitment and activation of eosinophils at the site of infection. As such this represents a novel mechanism of regulation of chemokine function in vivo. The existence of CCR3 ligands other than eotaxin (e.g., eotaxin-2) may reflect the evolution of host counter measures to parasite defense systems. (+info)
Regulation of endothelial heme oxygenase activity during hypoxia is dependent on chelatable iron.
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The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia. (+info)
Tyrosine phosphatase inhibitors selectively antagonize beta-adrenergic receptor-dependent regulation of cardiac ion channels.
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beta-Adrenergic receptor stimulation regulates the activity of several different cardiac ion channels through an adenylate cyclase/cAMP/protein kinase A-dependent mechanism. Previous work has suggested that basal tyrosine kinase activity attenuates the beta-adrenergic responsiveness of these cardiac ion channels, supporting the idea that tyrosine phosphorylation exerts an inhibitory effect at some point in the common signaling pathway. To determine which element in the beta-adrenergic pathway is regulated by tyrosine kinase activity, we studied the effects of various protein tyrosine phosphatase (PTP) inhibitors on the cAMP-dependent regulation of the L-type Ca(2+) current in guinea pig ventricular myocytes. Three such compounds, sodium orthovanadate, peroxovanadate, and bpV(phen), had no effect on the basal Ca(2+) current, yet each caused a pronounced inhibition of the Ca(2+) current stimulated by the beta-adrenergic receptor agonist isoproterenol. These observations are consistent with the idea that basal tyrosine kinase activity is capable of inhibiting beta-adrenergic responses. However, these PTP inhibitors had no effect on cAMP-dependent stimulation of the Ca(2+) current via activation of adenylate cyclase with forskolin or activation of H(2)-histaminergic receptors with histamine. These results are consistent with the idea that inhibition of PTP activity produces an inhibitory effect involving a tyrosine kinase-dependent mechanism acting selectively at the level of the beta-adrenergic receptor. This signaling mechanism does not seem to be linked to tyrosine kinase activity associated with insulin and insulin-like growth factor receptors, because acute exposure to agonists of these receptors did not inhibit isoproterenol regulation of the Ca(2+) current. (+info)
Mechanisms of DNA cleavage by copper complexes of 3-clip-phen and of its conjugate with a distamycin analogue.
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Mechanisms of DNA oxidation by copper complexes of 3-Clip-Phen and its conjugate with a distamycin analogue, in the presence of a reductant and air, were studied. Characterisation of the production of 5-methylenefuranone (5-MF) and furfural, associated with the release of nucleobases, indicated that these copper complexes oxidised the C1' and C5' positions of 2-deoxyribose, respectively, which are accessible from the DNA minor groove. Oxidation at C1' was the major degradation route. Digestion of DNA oxidation products by P1 nuclease and bacterial alkaline phosphatase allowed characterisation of glycolic acid residues, indicating that these copper complexes also induced C4' oxidation. However, this pathway was not associated with base propenal release. The ability of the copper complex of the 3-Clip-Phen conjugate with the distamycin analogue to produce sequence-selective DNA cleavage allowed confirmation of these mechanisms of DNA oxidation by PAGE. Comparison of DNA cleavage activity showed that conjugation of 3-Clip-Phen with a DNA minor groove binder, like the distamycin analogue, decreased both its ability to perform C1' oxidation as well as the initial rate of the reaction, but this conjugate is still active after 5 h at 37 degrees C, making it an efficient DNA cleaver. (+info)
The RepE initiator is a double-stranded and single-stranded DNA-binding protein that forms an atypical open complex at the onset of replication of plasmid pAMbeta 1 from Gram-positive bacteria.
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The RepE protein of the broad host range pAMbeta1 plasmid from Gram-positive bacteria is absolutely required for replication. To elucidate its role, we purified the protein to near homogeneity and analyzed its interactions with different nucleic acids using gel retardation assays and footprinting experiments. We show that RepE is monomeric in solution and binds specifically, rapidly, and durably to the origin at a unique double-stranded binding site immediately upstream from the initiation site of DNA replication. The binding induces only a weak bend (31 degrees ). Unexpectedly, RepE also binds nonspecifically to single-stranded DNA with a 2-4-fold greater affinity than for double-stranded origin. On a supercoiled plasmid, RepE binding to the double-stranded origin leads to the denaturation of the AT-rich sequence immediately downstream from the binding site to form an open complex. This open complex is atypical since (i) its formation requires neither multiple RepE binding sites on the double-stranded origin nor strong bending of the origin, (ii) it occurs in the absence of any cofactors (only RepE and supercoiling are required), and (iii) its melted region serves as a substrate for RepE binding. These original properties together with the fact that pAMbeta1 replication depends on a transcription step through the origin on DNA polymerase I to initiate replication and on a primosome to load the replisome suggest that the main function of RepE is to assist primer generation at the origin. (+info)
Streptolysin O-permeabilized granulocytes shed L-selectin concomitantly with ceramide generation via neutral sphingomyelinase.
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Cleavage of membrane-associated L-selectin regulates leukocyte rolling on vascular endothelium at sites of inflammation. We report that rapid and massive shedding of L-selectin occurs from granulocytes attacked by the pore-forming bacterial toxin streptolysin O (SLO). Shedding was not induced by an SLO mutant that retained binding capacity but lacked pore-forming activity. Cells permeabilized with SLO exhibited a 1.5-fold increase in the activity of neutral sphingomyelinase, which was accompanied by increased ceramide formation. L-selectin cleavage was inducible by treatment of cells with bacterial sphingomyelinase, and also through exogenous application of a cell-permeable ceramide analog. Our data identify a novel path to the shedding process and show that activation of neutral sphingomyelinase with the generation of ceramide is an important event underlying enhanced sheddase function in cells permeabilized by a pore-forming toxin. (+info)