Enterococcus hirae vacuolar ATPase is expressed in response to pH as well as sodium

AbstractThe Enterococcus hirae ntp operon encodes both a vacuolar ATPase, which transports Na+ as well as Li+, and the KtrII K+ transporter. A plasmid, in which the chloramphenicol acetyltransferase gene (CAT) was placed downstream of the ntp promoter, was introduced into a mutant totally defective in Na+ extrusion. The CAT activity of this transformant was increased preferentially by addition of NaCl, but not by LiCl, in the media or by elevating the medium pH, correlating well with the increase in amounts of the ATPase subunits observed by Western blotting. The physiological significance of these responses of the ntp promoter is discussed

N 1 -Dansyl-Spermine and N 1 -(n-Octanesulfonyl)-Spermine, Novel Glutamate Receptor Antagonists: Block and Permeation of N-Methyl-D-Aspartate Receptors

SUMMARY The effects of several N-sulfonyl-polyamines, including N 1 -dansyl-spermine (N 1 -DnsSpm) and N 1 -(n-octanesulfonyl)-spermine (N 1 -OsSpm), were studied at recombinant N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus laevis oocytes. N 1 -DnsSpm and N 1 -OsSpm inhibited NMDA receptors and were ϳ1000-fold more potent than spermine in oocytes voltage-clamped at Ϫ70 mV. Block by N 1 -DnsSpm and N 1 -OsSpm was strongly voltage dependent, being more pronounced at hyperpolarized membrane potentials. With the Woodhull model of voltage-dependent channel block, the values of K d (0) were 779 M, 882 M, and 7.4 mM and those of z␦ were 2.58, 2.57, and 1.07 for N 1 -DnsSpm, N 1 -OsSpm, and spermine, respectively. This suggests that an increase in the voltage dependence of block together with an increase in affinity contributes to the increased potencies of N 1 -DnsSpm and N 1 -OsSpm compared with spermine. Sensitivity to N 1 -DnsSpm was reduced by mutation NR1(N616Q) and was increased by mutations NR1(N616G) and NR2A(N615G). The NR1(N616G) and NR2A(N615G) mutations decreased the K d (0) value of N 1 -DnsSpm without affecting z␦, whereas the NR1(N616Q) mutation reduced z␦. These mutations may alter the accessibility of part of the polyamine binding site within the channel pore or directly alter the properties of that site. Block by N 1 -DnsSpm (0.3 M) was almost complete at Ϫ100 mV, and there was no relief of block at extreme negative membrane potentials (Ϫ100 to Ϫ200 mV) at wild-type NR1/NR2A channels. In contrast, block by N 1 -DnsSpm was partially relieved at extreme negative potentials at receptors containing NR1(N616G) or NR2A(N615G), suggesting that N 1 -DnsSpm can permeate these mutant channels but not wild-type NR1/NR2A channels. This is hypothesized to be due to an increase in the pore size of channels containing NR1(N616G) or NR2A(N615G), which allows passage of the bulky head group of N 1 -DnsSpm. In contrast to N 1 -DnsSpm, N 1 -OsSpm could easily permeate wildtype NR1/NR2A channels, presumably because the head group of N 1 -OsSpm can pass through the narrowest part of the channel pore. N-Sulfonyl-polyamines such as N 1 -DnsSpm and N 1 -OsSpm represent a new class of polyamine antagonists with which to study glutamate receptor ion channels. The endogenous polyamine spermine has a variety of effects on NMDA and non-NMDA glutamate receptors (1, 2). At NMDA receptors, spermine has both stimulatory and inhibitory effects when applied extracellularly (3-7). Inhibition of NMDA receptors by spermine is strongly voltage dependent and may be caused by an open-channel block and/or screening of surface charges around the mouth or vestibule of the ion channe

Molecular Characteristics of Toxicity of Acrolein Produced from Spermine

Acrolein (CH2=CH-CHO), an unsaturated aldehyde produced from spermine, is one of the major contributors to oxidative stress. Acrolein has been found to be more toxic than reactive oxygen species (H2O2 and •OH), and it can be easily conjugated with proteins, bringing about changes in nature of the proteins. Acrolein is detoxified by glutathione in cells and was found to be mainly produced from spermine through isolating two cell lines of acrolein-resistant Neuro2a cells. The molecular characteristics of acrolein toxicity and tissue damage elicited by acrolein were investigated. It was found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH); cytoskeleton proteins such as vimentin, actin, α- and β-tubulin proteins; and apolipoprotein B-100 (ApoB100) in LDL are strongly damaged by acrolein conjugation. In contrast, activities of matrix metalloproteinase-9 (MMP-9) and proheparanase (proHPSE) are enhanced, and antibody-recognizing abilities of immunoglobulins are modified by acrolein conjugation, resulting in aggravation of diseases. The functional changes of these proteins by acrolein have been elucidated at the molecular level. The findings confirmed that acrolein is the major contributor causing tissue damage in the elderly

Molecular Characteristics of Toxicity of Acrolein Produced from Spermine

Acrolein (CH2=CH-CHO), an unsaturated aldehyde produced from spermine, is one of the major contributors to oxidative stress. Acrolein has been found to be more toxic than reactive oxygen species (H2O2 and •OH), and it can be easily conjugated with proteins, bringing about changes in nature of the proteins. Acrolein is detoxified by glutathione in cells and was found to be mainly produced from spermine through isolating two cell lines of acrolein-resistant Neuro2a cells. The molecular characteristics of acrolein toxicity and tissue damage elicited by acrolein were investigated. It was found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH); cytoskeleton proteins such as vimentin, actin, α- and β-tubulin proteins; and apolipoprotein B-100 (ApoB100) in LDL are strongly damaged by acrolein conjugation. In contrast, activities of matrix metalloproteinase-9 (MMP-9) and proheparanase (proHPSE) are enhanced, and antibody-recognizing abilities of immunoglobulins are modified by acrolein conjugation, resulting in aggravation of diseases. The functional changes of these proteins by acrolein have been elucidated at the molecular level. The findings confirmed that acrolein is the major contributor causing tissue damage in the elderly