Arachidonic acid activatable electrogenic H+ transport in the absence of cytochrome b558 in human T lymphocytes

Káldi, Krisztina; Szászi, Katalin; Koncz, Gábor; Suszták, Katalin; Ligeti, Erzsébet

doi:10.1016/0014-5793(96)00105-6

Cited by 14 publications

(8 citation statements)

References 30 publications

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“…The blots were stained with Fast Green (Sigma, Buchs, Switzerland) to visualize total proteins [Luo et al, 2006]. Western blotting was performed as described previously [Kaldi et al, 1996] with anti-p22 phox antibody 1:1,000 (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were visualized using enhanced chemiluminescence (ECL) reagent and X-ray film.…”

Section: Western Blot Assessment Of P22 Phox Expression In Variant Cementioning

confidence: 99%

Three common polymorphisms in theCYBAgene form a haplotype associated with decreased ROS generation

et al. 2009

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NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation.

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Section: Western Blot Assessment Of P22 Phox Expression In Variant Cementioning

confidence: 99%

Three common polymorphisms in theCYBAgene form a haplotype associated with decreased ROS generation

et al. 2009

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“…In this study we demonstrate for the first time the presence of voltage‐activated proton currents in human leukaemic Jurkat T cells and in human peripheral blood B and T lymphocytes. The existence of H + channels in pig and human lymphocytes was proposed previously on the basis of arachidonic acid‐stimulated, Zn 2+ ‐sensitive pH changes (Káldi et al 1994, 1996). In fact, if one accepts the proposal that the gp91 phox component of NADPH oxidase is itself a voltage‐gated proton channel (Henderson et al 1995), then B lymphocytes must express these channels, because B lymphocytes and B lymphocyte‐derived cell lines can generate the superoxide anion, O − 2 , evidently by an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase similar to that in phagocytes (Volkman et al 1984; Maly et al 1988; Hancock et al 1990; Leca et al 1990; Leca et al 1991).…”

mentioning

confidence: 97%

Voltage‐activated proton currents in human lymphocytes

Schilling

Gratopp

DeCoursey

et al. 2002

The Journal of Physiology

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Journal of PhysiologyJurkat cells (Benichou et al. 1989;Gulbins et al. 1996), but sparingly expressed in T lymphocytes which lack this capability (van Reyk et al. 2001). We show here that the levels of H + channel expression in human B lymphocytes and in Jurkat cells are quantitatively more than adequate to sustain NADPH oxidase activity in these cells. METHODS CellsJurkat T cells. The human T leukaemia cell line Jurkat E6-1 was obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Jurkat T cells were grown in RPMI 1640 medium (GibcoBRL, Eggenstein, Germany) supplemented with 10 % fetal bovine serum and 2 mM glutamine at densities of 1-9 w 10 5 in a 37°C humidified incubator with 5 % CO 2 , and split three times weekly.Human lymphocytes. Venous blood was drawn from healthy adult volunteers after informed written consent was obtained according to procedures approved by the local Institutional Review Board and in accordance with Federal regulations. Lymphocytes were obtained from the whole blood of the donors. The suspension was layered on top of ficoll-hypaque (AmershamPharmarcia, Freiburg, Germany) after dilution with an equal volume of RPMI 1640 (GibcoBRL, Eggenstein, Germany). After centrifugation (30 min, 500 g, room temperature), mononuclear cells were harvested from the interphase, washed twice with lowspeed centrifugation (200 g, 10 min, 20°C) to remove platelets, and resuspended at 1.5 w 10 8 ml _1 in degassed RPMI1640 containing 0.5 % bovine serum albumin and 2 mM EDTA. B cells were separated by negative depletion using the MACS B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. Briefly, after blocking Fc receptors with aggregated human immunoglobulin, cells were incubated with murine hapten-conjugated monoclonal antibodies directed against human CD2, CD4, CD11b, CD16, CD36 and anti-IgE at 10°C for 10 min, washed twice, and then incubated with anti-hapten microbeads at 10°C for 15 min. The washed cell suspension was then placed on a LS+/VS+ column (Miltenyi), and the unlabelled cells passing through were collected from the effluent. This fraction consisted of 97 % CD19+ cells, as judged by flow cytometry after staining with fluoresceinisothiocyanate-conjugated Leu-12 monoclonal antibody (Becton Dickinson, Heidelberg, Germany). Subsequently, the LS+/VS+ column was placed outside the magnetic field, and the retained cells were eluted. This B cell-depleted fraction contained 80-90 % of CD3+ cells as judged by flow cytometry after staining with fluoresceinisothiocyanate-conjugated anti-CD3 monoclonal antibody (Dako, Hamburg, Germany).

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“…Expression. Voltage-activated proton currents were detected in human tonsillar T lymphocytes (68) and in human peripheral blood T lymphocytes (126). The expression of HVCN1 mRNA and H V 1 protein by mouse T lymphocytes has been recently confirmed (123).…”

Section: T Lymphocytesmentioning

confidence: 86%

Voltage-Gated Proton Channels as Novel Drug Targets: From NADPH Oxidase Regulation to Sperm Biology

Seredenina

Demaurex

Krause

2015

Antioxidants & Redox Signaling

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Arachidonic acid activatable electrogenic H⁺ transport in the absence of cytochrome b₅₅₈ in human T lymphocytes

Cited by 14 publications

References 30 publications

Three common polymorphisms in theCYBAgene form a haplotype associated with decreased ROS generation

Three common polymorphisms in theCYBAgene form a haplotype associated with decreased ROS generation

Voltage‐activated proton currents in human lymphocytes

Voltage-Gated Proton Channels as Novel Drug Targets: From NADPH Oxidase Regulation to Sperm Biology

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