Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).     

Identification of K(+) channels in the plasma membrane of maize subsidiary cells

The stomatal complex of Zea mays consists of two guard cells with the pore in between them and two flanking subsidiary cells. Both guard cells and subsidiary cells are important elements for stoma physiology because a well-coordinated transmembrane shuttle transport of potassium and chloride ions occurs between these cells during stomatal movement. To shed light upon the corresponding transport systems from subsidiary cells, subsidiary cell protoplasts were enzymatically isolated and in turn, analyzed with the patch-clamp technique. Thereby, two K(+)-selective channel types were identified in the plasma membrane of subsidiary cells. With regard to their voltage-dependent gating behavior, they may act as hyperpolarization-dependent K(+) uptake and depolarization-activated K(+) release channels during stomatal movement. Interestingly, the K(+) channels from subsidiary cells and guard cells similarly responded to membrane voltage as well as to changes in the K(+) gradient. Further, the inward- and outward-rectifying K(+) current amplitude decreased upon a rise in the intracellular free Ca(2+) level from 2 nM to the micro M-range. The results indicate that the plasma membrane of subsidiary cells and guard cells has to be inversely polarized in order to achieve the anti-parallel direction of K(+) fluxes between these cell types during stomatal movement.     

In vitro antioxidant activity of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a vitamin E metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of alpha-CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS.+) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, alpha-tocopherol, ascorbic acid, and (-)-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, alpha-CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that alpha-CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na(+)/K(+)-ATPase

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.     

Application of the C4'-alkylated deoxyribose primer system (CAPS) in allele-specific real-time PCR for increased selectivity in discrimination of single nucleotide sequence variants

This study describes a quantitative real-time PCR-based approach for discrimination of single nucleotide sequence variants, called CAPS (C4' alkylated primer system). To increase the discrimination potential of DNA polymerases against competing sequence variants of single nucleotides, 3'-terminally modified primers were designed carrying a methyl residue bound to the C4' of the thymidylate deoxyribose. In a model sequence system positional dependencies of modified thymidylate (at -1, -2, -3) were tested for their influence on discrimination. Highest discrimination factors were obtained with the modification at the ultimate 3'-position. In a comparison between Taq and Pwo DNA polymerases, substantial better results were obtained by Taq DNA polymerase. In contrast to conventional PCR methods for discrimination of sequence variants, achieving a maximum discrimination potential of about 20, CAPS is capable of obtaining sequence-specific amplifications of a desired target among discriminated templates with a dynamic range of 1:100. Therefore, CAPS is a method able to quantitatively discriminate two sequence variants only differing in a single base (e.g., SNP alleles or point mutations). The range of applications of this easy to perform, fast and reliable technique reaches from medical diagnostics, transplantation medicine, molecular and cell biology to human genetics. Targeting of SNPs assures a universal exertion of this method, since these markers are gender-independent, highly abundant and ubiquitous.     

Cloning and expression of the coding regions of the heat shock proteins HSP10 and HSP16 from Piscirickettsia salmonis

The genes encoding the heat shock proteins HSP10 and HSP16 of the salmon pathogen Piscirickettsia salmonis have been isolated and sequenced. The HSP10 coding sequence is located in an open reading frame of 291 base pairs encoding 96 aminoacids. The HSP16 coding region was isolated as a 471 base pair fragment encoding a protein of 156 aminoacids. The deduced aminoacid sequences of both proteins show a significant homology to the respective protein from other prokaryotic organisms. Both proteins were expressed in E. coli as fusion proteins with thioredoxin and purified by chromatography on Nicolumn. A rabbit serum against P. salmonis total proteins reacts with the recombinant HSP10 and HSP16 proteins. Similar reactivity was determined by ELISA using serum from salmon infected with P. salmonis. The possibility of formulating a vaccine containing these two proteins is discussed.