Inhibition of enzymes and oxidative damage of red blood cells induced by t-butylhydroperoxide-derived radicals

The effects of t-butylhydroperoxide (tBHP), its alkoxyl radical (tBuO.) and its peroxyl radical (tBuOO.) in model systems and on red blood cells were studied. Glyceraldehyde-3-phosphate dehydrogenase was strongly inhibited by tBHP via a direct reaction of the hydroperoxide with an essential sulfhydryl group in the enzyme molecule. Several other enzymes were unaffected by tBHP. Alcohol dehydrogenase was strongly inhibited by tBuO. but was much less sensitive to tBuOO.. Lysozyme, lactate dehydrogenase and trypsin, on the other hand, were very sensitive to the peroxyl and not, or much less, to the alkoxyl radical, whereas acetylcholinesterase was very sensitive to both radicals. tBuOO. caused covalent binding of tryptophan, tyrosine, histidine and methionine to serum albumin. The corresponding alkoxyl radical was ineffective in this respect. Conversely, tBuO. caused peroxidation of linolenic acid, whereas tBuOO. did not. Incubation of human erythrocytes with tBHP caused lipid peroxidation and K+ leakage. Both effects were caused by tBHP-derived radicals generated in a reaction of the hydroperoxide with hemoglobin. With radical scavengers it was possible to dissociate tBHP-induced lipid peroxidation and K+ leakage, demonstrating that these two processes are not causally related. Experimental results indicate that tBuO. causes lipid peroxidation, whereas tBuOO. is responsible for K+ leakage.     

Plastids with densely staining thylakoid contents inNymphoides indica

Summary Most chloroplasts of the lower epidermal and sub-epidermal cells in mature floating leaves ofNymphoides indica have densely staining thylakoid loculi which contrast markedly with electron translucent loculi of chloroplasts elsewhere in the leaves. Examination of leaf primordia (< 1 mm long) showed that granal membranes arise within the plastid stroma and already at that stage the stainable substance can be seen associated with the lamellae. In slightly enlarged primordia (5 mm long), nearly all plastids of the young leaf contain thylakoids with stained loculi, but by the time the leaf reaches 25 mm in length, the staining is restricted to the lower epidermal layers.     

Adaptation of transient responses of a movement-sensitive neuron in the visual system of the blowfly Calliphora erythrocephala

We studied temporal response properties of the H1 neuron by extracellular recording. This neuron is a wide-field movement-sensitive element in the visual system of the blowfly (Calliphora erythrocephala). If the neuron is stimulated with a stepwise pattern displacement in its preferred direction, it responds with a burst of action potentials. By repeating the stimulus step one obtains the average of the step response: a 20ms latency time followed by a sharp increase in average firing rate and a slower decay to the resting activity. We report that the characteristic decay time of the step response depends on the stimulus history. If the stimulus moved prior to the step, the higher the pattern velocity, the faster was the decay of the step response to the resting level. In quantitative terms, for velocities in the range 0.4–100°/s, the decay time-constant varies from 300–10ms and is smaller for higher velocities. The time-constant is only weakly affected by other stimulus parameters such as modulation depth or spatial wavelength, and is set independently in different areas of the visual field where it is tuned to the local velocity. We discuss a possible advantage of this form of adaptation for the processing of visual signals: The performance of the nolinear operations that extract information from the visual input can be optimized by prefiltering signals in the individual visual columns with a time-constant that decreases with stimulus velocity. It will be shown that both the test step response and the response to continuous movement can be described reasonably well by a correlation model with input filters that adapt their time-constants.     

The role of ATP in the control of H+-galactoside symport in the yeast Kluyveromyces marxianus.

Transport of methyl beta-D-thiogalactoside and p-nitrophenyl beta-D-galactoside is shown to proceed through the H+-lactose symporter of Kluyveromyces marxianus. Uptake of these compounds is strongly reduced under anaerobic conditions or aerobically in the presence of antimycin. It is shown that antimycin treatment affects p-nitrophenyl beta-D-galactoside uptake in a similar way as it affects the cellular amount of ATP, suggesting regulation of p-nitrophenyl beta-D-galactoside transport by ATP. Also, manipulation of cellular ATP by antimycin treatment followed by glucose incubation, or by aerobic incubation of cells with 2-deoxy-D-glucose, showed a similar dependence of galactoside uptake on the ATP level. Transport of the lipophilic cation tetraphenylphosphonium is affected by ATP variations in a similar way as galactoside influx. It is concluded that ATP regulates H+-galactoside symport by its influence on charge translocation. It is discussed that a membrane ATPase probably plays a central role in the control of the activity of H+-sugar symport.     

Toxic effects of ozone on murine L929 fibroblasts. Damage to DNA.

Damage to DNA caused by exposure of L929 fibroblasts to ozone was reflected by the generation of strand breaks, DNA inter-strand cross-links and DNA-protein cross-links. Addition of propan-2-ol, a hydroxyl radical scavenger, did not affect the formation of strand breaks. In model experiments it appeared that both purines and pyrimidines were involved in DNA inter-strand and DNA-protein cross-links.     

Kinetic analysis of L-lactate transport in human erythrocytes via the monocarboxylate-specific carrier system

Three parallel pathways of L-lactate transport across the membrane of human red blood cells can be discriminated: (a) by nonionic diffusion; (b) via the band 3 anion exchange protein; and (c) via a specific monocarboxylate carrier system. Influx of lactate via the latter system leads to alkalinization of the medium, suggesting lactate-proton symport. Kinetic analysis of initial lactate influx via the monocarboxylate carrier indicates a symport system with ordered binding of the two ligands, in the sense that a proton binds first to the translocator, followed by lactate binding to the protonated carrier. The influence of varying trans-pH under conditions of net (zero-trans) flux with constant cis-pH indicates that the monocarboxylate translocator should be considered as a mobile carrier, with the ligand-binding sites exposed alternatively to the outside and the inside of the membrane.     

Sugar Transport and Metal Binding in Yeast

The uptake of sugars by yeast can be separated into two classes. The first involves the uptake of sorbose or galactose by starved cells, and the uptake of glucose by iodoacetate-poisoned cells. These uptakes do not involve any changes in Ni⁺⁺- or Co⁺⁺-binding by the cell surface, are not inhibited by Ni⁺⁺, are inhibited by UO₂ ⁺⁺ in relatively high concentrations, are characterized by high Michaelis constants and low maximal rates and by a final equilibrium distribution of the sugars. The second involves the uptake of glucose in unpoisoned cells and galactose in induced cells. These uptakes are characterized by a reduction of Ni⁺⁺- and Co⁺⁺-binding, by a partial inhibition by Ni⁺⁺, by an inhibition with UO₂ ⁺⁺ in relatively low concentrations, and by a low Km and a high Vm. In the case of galactose in induced cells, previous studies demonstrate that the sugar is accumulated against a concentration gradient. It is suggested that the first class of uptakes involves a "facilitated diffusion" via a relatively non-specific carrier system, but the second represents an "uphill" transport involving the highly specific carriers, and phosphoryl groups (cation-binding sites) of the outer surface of the cell membrane.     

Effects of semicarbazide on oxidative processes in human red blood cell membranes

Semicarbazide can interfere with oxidative processes in the red blood cell membrane via different modes of action.Treatment of human red blood cell membranes with O₃ results, among other effects, in cross-linking of membrane proteins and inhibition of glyceraldehyde-3-phosphate dehydrogenase activity. Semicarbazide inhibits these effects by acting as an O₃ scavenger. The effect of semicarbazide as an O₃ scavenger is complicated by the fact that ozonolysis of semicarbazide yields a product that causes inhibition of glyceraldehyde-3-phosphate dehydrogenase.Glyceraldehyde-3-phosphate dehydrogenase inhibition can also be provoked by incubation of membrane suspensions with O₃-treated phospholipids. Semicarbazide prevented this effect by interaction with an inhibitory O₃-phospholipid reaction product.Protoporphyrin-induced photodynamic cross-linking of membrane proteins is chemically distinct from O₃-induced cross-linking. Photodynamic cross-linking is also inhibited by semicarbazide, in this case via reaction with a histidine photooxidation product.     

Effects of phytoplankton on the elemental composition (C,N, P) of suspended particulate material in the lower river Rhine

The spatial and temporal distribution of element concentrations were monitored together with chlorophyll a as an indicator of algal density to assess the effect of phytoplankton on the elemental composition (C, N, P) of suspended materials in the lower Rhine. The high concentrations of particulate C, N and P in the river were found to decrease in the delta and to increase again in the estuarine turbidity zone. Phytoplankton blooms increased the concentrations of particulate C, N, and P significantly in the upstream part of the river. In summer 1989, 15–65% of the particulate C and 20–75% of the particulate N were attributable to phytoplankton. Together with published data these observations indicate that in eutrophic rivers, the input of organic materials from the catchment is strongly modified and supplemented by in situ growth of phytoplankton. During seaward transport the phytoplankton and the particulate elements disappeared from the river water concomitantly with the suspended matter, indicating an increased retention of these elements due to sedimentation. In contrast, soluble ammonia, nitrite and phosphate increased in the tidal reaches of the river because of local input in the harbour and city of Rotterdam and because of mineralization. Therefore the total nutrient load of the Rhine estimated at the German/Dutch border does not reflect the actual input into the sea.     

Photodynamic effects of protoporphyrin on human erythrocytes. Nature of the cross-linking of membrane proteins

Protoporphyrin-sensitized photooxidation in human red blood cell membranes leads to severe deterioration of membrane structure and function. The membrane damage is caused by direct oxidation of amino acid residues, with subsequent cross-linking of membrane proteins. The chemical nature of these cross-links was studied in model systems, isolated spectrin and red cell ghosts. Cysteine and methionine are not involved in the cross-linking reaction. Further it could be shown that dityrosine formation, the crucial mechanism in oxidative cross-linking of proteins by peroxidase-H2O2 treatment, plays no role in photodynamic cross-linking. Experimental evidence indicated that a secondary reaction between free amino groups and a photooxidation product of histidine, tyrosine or tryptophan is involved in photodynamic cross-linking. This was deduced from the reaction observed between compounds containing a free amino group and photooxidation products of these amino acids, both in model systems, isolated spectrin and erythrocyte ghosts. In accordance, succinylation of free amino groups of membrane proteins or addition of compounds with free amino groups protected against cross-linking. Quantitative data and consideration of the reaction mechanisms of photodynamic oxidation of amino acids make it highly probable that an oxidation product of histidine rather than of tyrosine or tryptophan is involved in the cross-linking reaction, via a nucleophilic addition by free amino groups.