Eddy M. Massa

Epidermal Lignin Deposition in Quinoa Cotyledons in Response to UV-B Radiation¶

Abstract UV-B radiation (280–320 nm) is harmful to living organisms and has detrimental effects on plant growth, development and physiology. In this work we examined some mechanisms involved in plant responses to UV-B radiation. Seedlings of quinoa (Chenopodium quinoa Willd.) were exposed to variable numbers of UV-B radiation doses, and the effect on cotyledons was studied. We analyzed (1) cotyledons anatomy and chloroplasts ultrastructure; (2) peroxidase activity involved in the lignification processes; and (3) content of photosynthetic pigments, phenolic compounds and carbohydrates. Exposure to two UV-B doses induced an increase in the wall thickness of epidermal cells, which was associated with lignin deposition and higher activity of the peroxidase. The chloroplast ultrastructure showed an appearance typical of plants under shade conditions, likely in response to reduced light penetration into the mesophyll cells due to the screening effect of epidermal lignin deposition. Exposure to UV-B radiation also led to (1) enhancement in the level of phenolics, which may serve a protective function; (2) strong increase in the fructose content, a fact that might be related to higher requirement of erythrose-4P as a substrate for the synthesis of lignin and phenolics; and (3) reduction in the chlorophyll concentration, evidencing alteration in the photosynthetic system. We propose that the observed lignin deposition in epidermal tissues of quinoa is a resistance mechanism against UV-B radiation, which allows growing of this species in Andean highlands.

Hormone action and membrane fluidity: Effect of insulin and cortisol on the hill coefficients of rat erythrocyte membrane-bound acetyl-cholinesterase and (Na+ + K+)-ATPase

Summary The influence of insulin and cortisol on the Hill coefficients for the inhibition by F- of rat erythrocyte membrane-bound acetyl-cholinesterase and (Na+ + K+)-ATPase was studied “in vitro”. The study was carried out with erythrocyte membranes exhibiting a high or low fatty acid fluidity, which were obtained from rats fed a corn oil or lard supplemented diet, respectively. The hormone-induced changes in the Hill coefficients of both enzymes are interpreted to mean that insulin decreases and cortisol enhances membrane fluidity.

Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2☆

NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress. In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties. Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm. This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA. The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein. There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy. Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus. A NDH-2 topology model, based on the secondary structure prediction, is proposed. This is the first description of a copper-containing NADH dehydrogenase. Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.

Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in Escherichia coli

We are studying the action of tert-butylhydroperoxide (t-BOOH) on Escherichia coli as a model system for peroxide toxicity. In our previous report (De la Cruz-Rodriguez, L.C., Farías, R.N. and Massa, E.M. (1990) Biochim. Biophys. Acta 1015, 510-516), the respiratory chain was identified as a major target of t-BOOH. In the present paper, we study further the effect of t-BOOH on the NADH oxidase of the E. coli respiratory chain to clarify the mechanism of damage, especially regarding the identity and role of the metal ion involved. The results are: (a) t-BOOH toxicity is mediated by membrane-bound copper ions; (b) a small pool of the membrane-bound copper is reduced from Cu(II) to Cu(I) in the presence of NADH and other respiratory substrates (succinate, D-lactate); (c) this reduction of copper occurs at 37 degrees C but not at 0 degrees C or when the membranes are inactivated by previous heating; (d) the Cu(I) generated by reduction of Cu(II) during membrane preincubation with NADH, is oxidized by t-BOOH with simultaneous inactivation of the NADH oxidase, whereas treatment with only t-BOOH (without NADH) has no effect on the oxidase. It is concluded that the effect of t-BOOH on the respiratory chain is mediated by redox cycling of copper. It is proposed that the damage results from activation of the hydroperoxide through its interaction with Cu(I) in a site-specific Fenton-type reaction.

Quenching of bathocuproine disulfonate fluorescence by Cu(I) as a basis for copper quantification.

In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.

Specific localization of the respiratory alternative oxidase in meristematic and xylematic tissues from developing soybean roots and hypocotyls.

We used tissue printing and specific immunostaining to examine the localization of the alternative oxidase (AOX) protein in correlation with measurements of AOX capacity. Selected root and hypocotyl regions were analyzed during the first 14 d of growth. It is shown that AOX protein is localized in the apical meristem and in developing xylem. The temporal pattern of expression is coincident with the evolution of AOX capacity. Data suggest that AOX expression is linked to xylem differentiation. Since heat is a major product of the alternative pathway, we speculate that thermogenesis is implicated in morphogenesis.

Fatty acid dependent hydrogen peroxide production in lactobacillus

Lactobacillus leichmanii growing in complex medium supplemented with decanoic acid accumulated high concentrations of hydrogen peroxide in the culture. The H2O2-generating system was specifically induced by one of the saturated fatty acids from 4:0 to 16:0 or oleic acid. The induction of this system was associated with the presence of a fatty acyl-CoA-dependent H2O2-generating activity in the cell-free extracts. This activity is shown for the first time in a procaryote organism.

Effect of phospholipids, Triton X-100 and biological membranes on redox systems involving tetrazolium salt reduction. Its implications for the assay of enzymatic activities

Abstract The influence of phospholipids and Triton X-100 on the time course of chemical and enzyme-mediated reductions of a commonly used tetrazolium salt, MTT, was studied. MTT reduction was followed by the absorbance changes at 570 nm. With ascorbate as reducing agent, a 3-fold increase in the initial rates of the absorbance changes and a 24 % increase in the final absorbance values were observed in the presence of Triton X-100 micelles or phospholipid vesicles. The enzyme-mediated reduction of MTT with NADH generated by the NAD-dependent lactate dehydrogenase was also enhanced in the presence of Triton X-100, phospholipids or erythrocyte membranes. No enhancement was observed following the enzymatic generation of NADH at 340 nm in the absence of MTT. The above findings were interpreted as arising from: a) solubilization or reduced MTT in the detergent micelles or phospholipid vesicles which favors the redox reaction occurring in the aqueous fase, and b) changes in the spectral properties of reduced MTT in aqueous and lipid-like media.

Damage of Escherichia coli cells by t-butylhydroperoxide involves the respiratory chain but is independent of the presence of oxygen

The action of t-butylhydroperoxide (tBOOH) on Escherichia coli cells has been studied as a model system for organic peroxide toxicity. Exposure of E. coli cells to tBOOH led to progressive and irreversible impairment of the respiratory function, an effect which was dependent on the availability of substrate. The effect of tBOOH on growth of E. coli with different carbon sources and alternative terminal electron acceptors was investigated. It was found that the sensitivity of E. coli to tBOOH under diverse growth conditions implicating a functional respiratory chain was greater than when the bacterium grew by fermentation. Also the mutant E. coli SASX76, which requires exogenous 5-aminolevulinic acid to synthesize the cytochromes, was more resistant to tBOOH when lacking a functional respiratory chain. These data point to the respiratory chain as a major target in the in vivo action of tBOOH. Experiments with isolated membranes also showed a tBOOH-induced damage of the respiratory chain monitored by impairment of the NADH oxidase. The effect of tBOOH was produced even under anaerobiosis, indicating that development of cell damage was independent of oxygen and, therefore, that neither oxygen-derived radicals nor lipid peroxidation were involved.

Membrane cooperative enzymes: interplay of insulin, glucagon and epinephrine on rat erythrocyte acetylcholinesterase system.

Insulin, at physiological plasma levels, affects the cooperativity of membrane-bound enzymes [ 1,2]. This action was detected in vitro through changes in the Hill h coefficient of rat erythrocyte membranebound acetylcholinesterase and (Na’ + K’)-ATPase [ 11. and in Escherichia coli membrane-bound Ca’+ATPase [2]. Insulin decreased the values of h of erythrocyte acetylcholinesterase [l] and E. coli Ca”-ATPase [2] and enhanced it in the erythrocyte (Na’ t K’)-ATPase system [ 11. The correlation between the membrane fluidity and the h values of the former enzymes were positive whereas for the latter enzyme it was negative [3,4]. Based on these findings we postulated that insulin decreases membrane fluidity [ 1,2]. In general insulin modifies metabolic activities in an opposite manner to that induced by epinephrine or glucagon [5], so it was of interest to study the actions of these hormones on a membrane-bound cooperative system. The present report provides the first evidence of a hormonal interplay between insulin, epinephrine, and glucagon on rat erythrocyte membrane-bound acetylcholinesterase (EC 3.1 .1.7). Epinephrine and insulin showed opposite effects on the Hill coefficient. Insulin 10m9 M decreases the values of h, whereas epinephrine lo-’ M raises them. Glucagon lo-” M has a blocking action on the insulin effect. The interplay between insulin, glucagon and epinephrine is in agreement with current ideas about the actions of these hormones on metabolic fuels. Part of the present work was reported earlier in preliminary form [6].