Vitamin E at high doses improves survival, neurological performance, and brain mitochondrial function in aging male mice

Male mice receiving vitamin E (5.0 g alpha-tocopherol acetate/kg of food) from 28 wk of age showed a 40% increased median life span, from 61 +/- 4 wk to 85 +/- 4 wk, and 17% increased maximal life span, whereas female mice equally supplemented exhibited only 14% increased median life span. The alpha-tocopherol content of brain and liver was 2.5-times and 7-times increased in male mice, respectively. Vitamin E-supplemented male mice showed a better performance in the tight-rope (neuromuscular function) and the T-maze (exploratory activity) tests with improvements of 9-24% at 52 wk and of 28-45% at 78 wk. The rates of electron transfer in brain mitochondria, determined as state 3 oxygen uptake and as NADH-cytochrome c reductase and cytochrome oxidase activities, were 16-25% and 35-38% diminished at 52-78 wk. These losses of mitochondrial function were ameliorated by vitamin E supplementation by 37-56% and by 60-66% at the two time points considered. The activities of mitochondrial nitric oxide synthase and Mn-SOD decreased 28-67% upon aging and these effects were partially (41-68%) prevented by vitamin E treatment. Liver mitochondrial activities showed similar effects of aging and of vitamin E supplementation, although less marked. Brain mitochondrial enzymatic activities correlated negatively with the mitochondrial content of protein and lipid oxidation products (r2 = 0.58-0.99, P < 0.01), and the rates of respiration and of complex I and IV activities correlated positively (r2 = 0.74-0.80, P < 0.01) with success in the behavioral tests and with maximal life span.     

Treatment of duodenal ulcers by antacid (Al-Mg-hydroxy-carbonate). A controlled, randomized, prospective, multicentre clinical trial

TISACID (a new, modern Hungarian Al-containing antacid) with a high acid-neutralizing capacity (greater than 26.8 mmol/g) also enhances gastric mucosal defense mechanisms (prostaglandin-dependent gastroprotection). A simple-blind, prospective, randomized, parallel multicentre clinical trial has been performed on both the clinical efficacy and possible side effects of TISACID monotherapy (Al-Mg-hydroxy-carbonate) on informed patients suffering from active duodenal ulcers. The study groups were as follows: Group "A": 3 g/day of TISACID (acid-neutralizing capacity = 78 mmol, n = 85), Group "B": 6 g/day of TISACID (acid-neutralizing capacity = 156 mmol, n = 88), Group "C": 12 g/day of TISACID (acid-neutralizing capacity = 312 mmol, n = 68), Group "D": (as control): (1.0 g/day cimetidine (HISTODIL, RGH, Budapest, n = 91). The total number of patients: 332. It was found that: 1. The new Hungarian antacid compound (both tablet and suspension) can essentially accelerate the healing rate of duodenal ulcers. 2. The cumulative healing rate of ulcers and the decrease of complaints can be achieved equally by relatively low doses of TISACID monotherapy and cimetidine alone. 3. There were no differences between the clinical potency and side-effects of TISACID tablet and suspension.     

Effect of the lipid interface on the catalytic activity and spectroscopic properties of a fungal lipase

Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.     

Impaired proteostasis contributes to renal tubular dysgenesis

Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell. The etiology of renal tubular dysgenesis (RTD) is linked to mutations in the angiotensin-converting enzyme (ACE). Here, we report the identification of a novel ACE mutation (Q1069R) in an RTD patient. ACE Q1069R is found sequestered in the endoplasmic reticulum and is also subject to increased proteasomal degradation, preventing its transport to the cell surface and extracellular fluids. Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels. In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.     

Derivation of a Bayes factor to distinguish between linked or pleiotropic quantitative trait loci

A simple procedure to calculate the Bayes factor between linked and pleiotropic QTL models is presented. The Bayes factor is calculated from the marginal prior and posterior densities of the locations of the QTL under a linkage and a pleiotropy model. The procedure is computed with a Gibbs sampler, and it can be easily applied to any model including the location of the QTL as a variable. The procedure was compared with a multivariate least-squares method. The proposed procedure showed better results in terms of power of detection of linkage when low information is available. As information increases, the performance of both procedures becomes similar. An example using data provided by an Iberian by Landrace pig intercross is presented. The results showed that three different QTL segregate in SSC6: a pleiotropic QTL affects myristic, palmitic, and eicosadienoic fatty acids; another pleiotropic QTL affects palmitoleic, stearic, and vaccenic fatty acids; and a third QTL affects the percentage of linoleic acid. In the example, the Bayes factor approach was more powerful than the multivariate least-squares approach.     

Factors shaping European rabbit abundance

This study analyses differences in European rabbitOryctolagus cuniculus (Linnaeus, 1758) abundance between fragmented and continuous populations and the relative importance of habitat structure (micro- and macrohabitat) and isolation in determining the abundance pattern in fragmented and continuous areas of central Spain. The species was mainly restricted to mediterranean vegetation habitat. In fragmented areas, rabbit abundance was linked to scrubland cover but was not correlated to pasture/cropland cover. The model explained very little of the observed variance. Distance to continuous populations did not improve the model. Stochastic phenomena or unmeasured factors (predation level, soil type) could be acting in this context. Rabbits were more abundant in continuous areas than in fragmented ones. In continuous areas, rabbit abundance was associated with mosaics of pastures, scrublands, and croplands. These habitat features are linked to shelter and feeding requirements of rabbits. The model explained an important part of the observed variance. This supports that management and conservation strategies should be based on the landscape pattern in each situation.     

Comparative study of electron mediators used in the electrochemical oxidation of NADH

A comparison of the behavior of six different redox mediators for the electrocatalysis of the oxidation of NADH, which are widely used in the construction of dehydrogenase-based biosensors, is reported. The redox mediators were, potassium hexacyanoferrate (II), Meldola's Blue (MB), dichlorophenolindophenol (DCPIP), p-benzoquinone (p-BQ), o-phenylenediamine (o-PDA) and 3,4-dihydroxybenzaldehyde (3,4-DHB). After incorporating each of them in the sensor system following four different strategies (in solution, entrapped in epoxy-composites, adsorbed or electropolymerized on the electrode surface), several aspects regarding repeatability and reproducibility were considered and compared.