Adverse immune reactions to gold. I. Chronic treatment with an Au(I) drug sensitizes mouse T cells not to Au(I), but to Au(III) and induces autoantibody formation

Upon weekly i.m. injections of disodium gold thiomalate (Na2AuTM) 100% of A.SW mice produced IgG autoantibodies to antinuclear Ag and nucleolar Ag, respectively; about 70% of C57BL/6 mice produced IgG antinuclear Ag, whereas DBA/2 mice were resistant. Moreover, C57BL/6 mice, but not DBA/2 mice, showed increased mesangial deposits of IgG. These alterations were due not to disodium thiomalate, but to the gold ion of Na2AuTM. An assumed T cell reactivity of susceptible mouse strains to Na2AuTM was tested by means of the direct popliteal lymph node (PLN) assay. However, no distinct PLN reaction to Na2AuTM was detectable. Likewise, AuCl did not induce a PLN reaction. Both Na2AuTM and AuCl contain gold in the Au(I) state. The poor PLN responses to Au(I) contrasted with the strong PLN responses to Au(III) compounds. PLN reactions to Au(III) were dose dependent, T cell dependent, and specific. When Au(III) was reduced to Au(I) by addition of Na2TM or methionine before testing in the PLN assay its sensitizing capacity was significantly decreased. Thus, the oxidation state of gold, i.e., Au(III) vs Au(I), plays a major role for its sensitizing capacity. Therefore, we propose that the Au(I) of Na2AuTM is oxidized to Au(III) before T cells are sensitized and adverse immunologic reactions develop. Results obtained with the adoptive transfer PLN assay indicated that, indeed, repeated i.m. injections of Na2AuTM sensitized A.SW and C57BL/6 splenic T cells to Au(III).     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Electrocatalytic properties of polypyrrole in amperometric electrodes

The electrocatalytic oxidation of NADH, ascorbate, urate, xanthine and H₂O₂ at different polypyrrole electrodes has been investigated. The conducting polymer was grown on platinum, glassy carbon, or graphite electrodes and modified by means of enclosed redox-active anions or other redox-active compounds covalently bound to either the N- or the β-position of the pyrrole. Copolymers of pyrrole and N-substituted pyrrole derivatives of chloranil or 2,3-dicholoro-1,4-naphthoquinone showed outstanding electrocatalytic properties for the oxidation of NADH. The application of these electrodes in amperometric steady-state measurements or flow-injection systems in combination with dehydrogenase reactions has been possible.     

Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells

Mono-, di-, and trisulfonic acids, including 4,4′-diacetamido stilbene-2,2′-disulfonic acid (DAS) and 2-(4′-amino phenyl)-6-methylbenzene thiazol-3′,7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluoro-benzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, 1974). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements. The effect of the irreversibly binding inhibitor 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS) on DNFB binding to the protein in band 3 was studied in an attempt to differentiate DNFB binding related to inhibition of anion permeability from DNFB binding which is not involved. At least three distinguishable populations of DNFB binding sites were found: (1) binding sites common for DNFB and SITS which are probably related to inhibition, (2) other common sites which are not related to inhibition and (3) different sites whose dinitrophenylation is not affected by SITS. The number of sites in population (1) was estimated to be 0.8–1.2 ± 10⁶/cell. A study of the concentration dependence of the inhibition of anion equilibrium exchange with 4,4′-isothiocyanato-2,2′-stilbene disulfonic acid (DIDS) and APMB further suggests that among the sites in population (1) a major fraction is susceptible to modification by APMB and DIDS while the rest is only susceptible to DIDS. It remains undecided whether these differences of susceptibility reflect differences of accessibility or reactivity.     

Ni‐Metalloid (B,Si, P,As, and Te) Alloys as Water Oxidation Electrocatalysts

Breakthroughs toward effective water‐splitting electrocatalysts for mass hydrogen production will necessitate material design strategies based on unexplored material chemistries. Herein, Ni‐metalloid (B, Si, P, As, Te) alloys are reported as an emergent class of highly promising electrocatalysts for the oxygen evolution reaction (OER) and insight is offered into the origin of activity enhancement on the premise of the surface electronic structure, the OER activation energy, influence of the guest metalloid elements on the lattice structure of the host metal (Ni), and surface‐oxidized metalloid oxoanions. The metalloids modify the lattice structure of Ni, causing changes in the nearest Ni–Ni interatomic distance (d_Ni–Ni). The activation energy E_a scales with d_Ni–Ni indicating an apparent dependence of the OER activity on lattice properties. During the OER, surface Ni atoms are oxidized to nickel oxyhydroxide, which is the active state of the catalyst, meanwhile, the surface metalloids are oxidized to the corresponding oxoanions that affect the interfacial electrode/electrolyte properties and hence the adsorption/desorption interaction energies of the reacting species.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

Amperometric enzyme biosensors based on optimised electron-transfer pathways and non-manual immobilisation procedures

Development of reagentless biosensors implies the tight and functional immobilisation of biological recognition elements on transducer surfaces. Specifically, in the case of amperometric enzyme electrodes, electron-transfer pathways between the immobilised redox protein and the electrode surface have to be established allowing a fast electron transfer concomitantly avoiding free-diffusing redox species. Based on the specific nature of different redox proteins and non-manual immobilisation procedures possible biosensor designs are discussed, namely biosensors based on (i) direct electron transfer between redox proteins and electrodes modified with self-assembled monolayers; (ii) anisotropic orientation of redox proteins at monolayer-modified electrodes; (iii) electron-transfer cascades via redox hydrogels; and (iv) electron-transfer via conducting polymers.     

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4 ^−/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4 ^−/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.