Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Timemresolved fluorescence of S-100a protein: effect of Ca, Mg and unilamellar vesicles of egg phosphatidylcholine

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca²⁺-binding protein S-100a both in the absence and in the presence of Ca²⁺ and/or Mg²⁺. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca²⁺, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg²⁺, the Ca²⁺ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca²⁺, this value was increased to 16.08 ns. In the presence of Mg²⁺, Ca⁺² increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg²⁺ indicate some interaction between lipids and S-100, likely mediated by this ion.     

cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

Interaction of trace elements in a longitudinal study of human milk from full-term and preterm mothers

Concentrations of 8 trace elements (Fe, Cu, Zn, Se, Br, Pb, Rb, and Sr) at different lactation time were measured by the PIXE multi-elemental technique. Time dependence and interelement correlations were studied. A total of 200 milk samples from 32 lactating mothers were supplied from 2 to 120 d after delivery of 26 full-term and 6 preterm infants. All elements showed a lognormal frequency-distribution. The Fe, Cu, Zn, and Se contents in preterm milk were found to be somewhat different with respect to full-term milk. Cu, Zn, Se, Br, Pb, and Rb concentrations declined with lactation time, both in pre- and full-term samples. Sr and Fe contents did not show any change with time. Detailed analysis of data by partial correlation and multiple regression methods was performed. No substantial differences between preterm and full-term samples were found in the results of partial correlation analysis. Cu and Zn were found to be correlated with lactation time, whereas the measured time dependence for the other elements has to be attributed to the effect of the existing interelement correlation. All the measured elements appeared to be correlated with at least one other element. In particular, Se was inversely correlated with Zn and directly with Cu. The zinc and copper contents in milk can therefore depend on the variation in the mother selenium intake.     

Genetic basis of sodium exclusion and sodium tolerance in yeast. A model for plants

A yeast strain carrying disruptions in TRK1 and ENA genes was very sensitive to Na⁺ because uptake discriminated poorly between K⁺ and Na⁺, and Na⁺ efflux was insignificant. Transformation with TRK1 and ENA1 restored discrimination, Na⁺ efflux and Na⁺ tolerance. Increasing external Ca²⁺ increased Na⁺ tolerance almost in the same proportion in TRK1 enal cells and in trkl ENAI cells, suggesting an unspecific effect of this cation. By using a vacuolar ATPase mutant, the role of the vacuole in Na⁺ tolerance was also demonstrated. The yeast model of Na⁺ exclusion and Na⁺ tolerance may be extended to plants.     

Ion-pair mechanism in square planar substitution. Reactivity of cationic platinum (II) complexes with negatively charged nucleophiles in solvents of high, medium and low polarity

The rates of displacement of dimethyl sulfoxide from the cation [Pt(phen) (CH₃) (Me₂SO)]⁺ by a series of uncharged and negatively charged nucleophiles have been measured in a methanol/water (19:1 vol./vol.) mixture. The starting complex and the reaction products were characterized either as solids or in solution by their IR and ¹H NMR spectra. The substitution reactions take place by way of a direct bimolecular attack of the ligand on the substrate. The sequence of reactivity observed is as expected on the basis of a nucleophilicity scale relevant for + 1 charged substrates ([Pt(en) (NH₃)Cl]⁺ used as standard). The difference of reactivity between the first (t-BuNH₂) and the last (SeCN⁻) members of the series spans five orders of magnitude. The value measured for the nucleophilic discrimination (1.55) is the highest found so far for cationic substrates. This is a result of the easy transfer of some of the electron density brought in by the incoming ligand into the ancillary ligands. When the reaction is carried out in a series of protic and dipolar aprotic solvents, using chloride ion as nucleophile, the rate of formation of [Pt (phen) (CH₃)Cl] is dominated by the extent of solvation of Cl⁻, as measured by its values of the Gibbs molar energy of transfer Δ_tG⁰. Conductivity measurements at 25°C in dichloromethane were fitted to the Fuoss equation and the values of the dissociation constants K_d for the ion pairs were calculated as follows: 2.27 × 10⁻⁵ M for Bu₄NCl, 2.75 × 10⁻⁵ M for Bu₄NSCN and 17.05 × 10⁻⁵ M for [Pt(phen) (CH₃) (Me₂SO)]PF₆. The pseudo-first-order rate constants k_obs for the reactions with Bu₄NCl, Bu₄NBr, Bu₄NSCN and Bu₄NI showed a curvilinear dependence on the concentration of the salt which levels off very soon (at concentrations higher than 0.005 M the kinetics are zero order in [Bu₄NX]). On addition of the inert electrolyte Bu₄NPF₆ the rates slow down and the kinetics follow the rate law k_obs = kK_ip[Bu₄NX]/[Bu₄NPF₆] + K_ip[Bu₄NX]). These findings fit well with a reaction scheme which involves a pre-equilibrium K_ip between ion pairs, followed by unimolecular substitution within the contact ion pair [Pt(phen) (CH₃) (Me₂SO)X]_ip. Values of the equilibrium constants K_ip for ion-pair exchange and of the internal substitution rates k were derived. The latter showed that the discrimination in reactivity between Cl⁻, Br⁻, SCN⁻ and I⁻ is greatly reduced with respect to aqueous solutions. The reason behind this may be desolvation of the ions coupled to the fact that a contact ion pair is already at a certain distance along the reaction coordinate in the direction of the transition state. Applications of the special salt effect and of ion pairing to synthesis are discussed.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.