Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

High dietary calcium level decreases colonic phytate degradation in pigs fed a rapeseed diet

The degradation of phytate (inositol hexaphosphate) in rapeseed meal diet not containing phytase activity was studied in 15 growing ileum-fistulated pigs. Stomach and small intestinal degradation and total gastrointestinal degradation were compared. The effect of addition of calcium carbonate to the rapeseed meal diet at two levels (9.2 and 18.5 g/kg diet) was investigated. A commercial barley-wheat-soybean diet with intrinsic phytase activity was used as reference. Phytate and its hydrolysis products in diets, ileal digesta and feces were determined by HPLC ion-pair chromatography. Hydrolysis of phytate in the stomach and small intestine was 35-45% in pigs fed the rapeseed meal diet independent of calcium addition, and 65% in pigs fed the reference diet. Total gastrointestinal degradation of phytate in pigs fed the rapeseed diet was 97, 77 and 42% (P < 0.001) when calcium intakes were 4.5, 9.9 and 15 g/d, respectively; total gastrointestinal degradation was 72% in pigs fed the reference diet. The intestinal phytate degradation pattern, when rapeseed diet was fed, indicated the activity of an unspecific phosphatase, whereas that of the reference diet indicated intrinsic dietary phytase activity. We conclude that dietary supplementation of calcium carbonate decreases the phytate degradation in the colon of pigs, but not in the stomach and small intestine.     

Acceptability of genetically modified cheese presented as real product alternative

European consumers, in general, have negative attitudes towards the use of gene technology in food production. The objective of this study was to examine whether taste and health benefits influence the acceptability of genetically modified (gm) products when they are presented as real product alternatives. Consumers in Denmark, Finland, Norway and Sweden (n=738) assessed two cheeses: one was labelled as genetically modified (preferred in an earlier product test) and the other as conventional (neutral in an earlier product test). A smaller control group received two cheeses with blind codes. Labelling decreased consumers' intentions to buy the originally preferred gm-labelled cheese, but still the intentions were at the same level with the conventionally labelled option. Participants chose two gm cheeses out of five possible when given the option to take cheese home after tasting. Intentions to buy gm cheese could best be explained by respondents' attitudes towards gene technology and perceived taste benefits. General health interest was also a reinforcer of intentions for gm cheese with reduced fat content.     

Student acceptance of unique meals

Students (N = 167) from the 4th, 8th, and 11th grades sorted meals, listed on individual cards, according to whether they would or would not eat them. Students were told to either consider eating the meals at home or at school. Forty-two different meals were selected from a pool of unique meals created by students in a previous study. The meals differed in head component, the presence or absence of vegetables, and the number of different foodstuffs. On average, approximately half of the meals were deemed acceptable, but older students accepted more meals than younger ones, boys accepted more meals than girls, and meals to be consumed at home were accepted more than those to be consumed at school. In addition, students tended to accept meals comprised of a small number of foodstuffs that did not contain vegetables. The also chose more meat-based meals over those containing fish or soup. These results suggest that students have rather unique food preferences that do not correspond well with nutritional standards. In order to address this problem we recommended that students be given more good choices in the school cafeteria through the introduction of a two-meal system and salad bars.     

The influence of NaCl and CO2 on the atmospheric corrosion of magnesium alloy AZ91

The influence of NaCl and CO₂ on the atmospheric corrosion of magnesium alloy AZ91 is studied in the laboratory. Samples were exposed under carefully controlled air and flow conditions; the relative humidity was 95%, the temperature was 22.0°C and the concentration of CO₂ was &amp;lt; 1 ppm or 350 ppm. Different amounts of sodium chloride (0–70 μg/cm²) were added before exposure. The corrosion products were analyzed by gravimetry, ion chromatography, X‐ray diffraction and scanning electron microscopy. Mass gain and metal loss results are reported. The combination of high humidity and NaCl is very corrosive towards AZ91. However, the NaCl‐induced corrosion is inhibited by ambient concentrations of CO₂. Exposure in the absence of CO₂ gives rise to heavy pitting, with brucite, Mg(OH)₂, being the dominant corrosion product. In the presence of CO₂ a layer of hydrated magnesium hydroxy carbonate, Mg₅(CO₃)₄(OH)₂ · 5 H₂O forms. A tentative corrosion mechanism is presented that explains the behavior in the two environments.     

Dissolution kinetics of ulexite in acetic acid solutions

Ulexite is an important boron mineral used for the production of boron compounds. The aims of this study are to examine the dissolution kinetics of ulexite in acetic acid solutions, and to present an alternative process to produce boric acid. In order to investigate the dissolution kinetics of ulexite in acetic acid solutions, the concentration of solution, reaction temperature, solid-to-liquid ratio, and particle size were selected as experimental parameters. It was determined that the dissolution rate of ulexite increased with increasing solution concentration and temperature and decreasing particle size and solid-to-liquid ratio. The activation energy of the process was found to be 55.8&amp;nbsp;kJ/mol.