Cellular response to oxidative stress at sulfhydryl group receptor sites on the erythrocyte membrane

Ellman's reagent was used to induce an oxidative stimulus on the exofacial membrane sulfhydryl groups of the human erythrocyte. Thiol-disulfide exchange occurring extracellularly was monitored using resonance Raman spectroscopy, and intracellular changes were observed by 1H spin echo nuclear magnetic resonance spectroscopy of the intact cell. The stimulus caused oxidation and depletion of the glutathione pool, which was followed at higher concentrations of Ellman's reagent by a depletion of intracellular ergothioneine levels. Larger changes are induced intracellularly than would be expected from the stoichiometry of the reaction at the exofacial surface. A mechanism is proposed which links exofacial sulfhydryl receptor sites via the transport proteins to spectrin and glutathione. The consequences for the cellular redox balance of an extracellular stimulus of this type are discussed.     

Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

The reduction of anti-tumour diaziridinyl benzoquinones

The properties of the semiquinone radicals produced for 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), have been investigated. AZQ semiquinone radicals can be produced from the reduction of AZQ by superoxide radicals, whereas BZQ semiquinone radicals are unstable in the presence of oxygen. The one-electron reduction potentials of the couples Q/Q-. at pH 7.0 were determined as -70 +/- 10 mV for AZQ and -376 +/- 15 mV for BZQ. The difference in these potentials is explained. As a consequence of ESR studies on the enzymatically produced radicals, we have considered the factors which determine the detection of ESR signals for reduced quinones produced in a biological system.     

Purification and properties of theDrosophila zen protein

Summary The zen protein is encoded by the zerknullt gene required for normal early development inDrosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40000. It binds specifically to a site about 900 bases upstream from thezen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.