Effects of HgCl2 on porphobilinogen-synthase (E.C. 4.2.1.24) activity and on mercury levels in rats exposed during different precocious periods of postnatal life

Porphobilinogen-synthase (PBG-synthase) is an enzyme extensively used as a bioindicator of metals and other oxidizing agents. The objective of this study was to verify the effects of HgCl(2) (5mg/kg/day, s.c.), a metal that mainly affects the nervous and renal systems, on kidney, liver and brain from rats exposed during one of the phases considered critical for development. Mercury decreased PBG-synthase activity from liver, kidney and brain and altered corporal, renal and cerebral weights. The kidney was the most sensitive tissue. It accumulated a large amount of metal and PBG-synthase activity was decreased up to 50%. The second period seemed to be the most sensitive, because in this phase the rats presented alterations in body, brain and kidney weights, and there was also an expressive inhibition in hepatic and renal PBG-synthase activities. In general, large quantities of metal accumulated in the tissues are in agreement with the inhibition verified in these tissues.     

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F₁F_O-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F₁F_O complex to work in the reverse mode, i.e. F₁-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F₁, Atp9p-ring, unassembled α-F₁ subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.     

Dopamine Neurotoxicity: Inhibition of Mitochondrial Respiration

Abstract: Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD₅₀ = 90 µg. Norepinephrine was less effective with LD₅₀ = 141 µg. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC₅₀ = 8 µ M , and that of norepinephrine was twice as much (IC₅₀ = 15 µ M ). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.     

Structural characteristics of the cold-water-soluble arabinoxylans from the white flour of the soft wheat variety Kadet

Cold-water-soluble arabinoxylan polymers from the soft wheat variety Kadet were isolated by a stepwise (NH₄)₂SO₄ precipitation and freed from co-precipitating proteins by pronase digestion. The purified arabinoxylans, representing 0·5% of the dry weight of the white flour, were fractionated by graded precipitation with ethanol. Monosaccharide analysis revealed the major carbohydrate fractions to contain arabinoxylans only. The arabinoxylans are heterogeneous in molecular size. Determination of the carbohydrate composition in conjunction with methylation analysis and ¹³C-NMR spectroscopy, indicated that they can be divided into specific groups, based on characteristic differences with respect to -xylopyranose to -arabinofuranose ratio and in 2,3,4-tri-:3,4-di-:4-mono-substituted -xylopyranose ratio.     

Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.     

Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis

We report the novel synthesis of cyclic PLP_139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H¹⁴⁷ replaces W¹⁴⁴ and F¹⁵¹-K¹⁵⁰ replace H¹⁴⁷ as TCR contacts, which may explain the difference on each peptide’s response.     

Mesenchymal stroma cells in peritoneal dialysis effluents from patients

Mesenchymal stroma cells (MSCs) have potential as an emerging cell therapy for treating many different diseases, but discovery of the practical sources of MSCs is needed for the large-scale clinical application of this therapy. This study was to identify MSCs in peritoneal dialysis (PD) effluents that were discarded after PD. The effluents were collected from patients who were on the dialysis for less than 1 month. Adherent cells from the effluents were isolated by incubation in serum-containing medium in plastic culture dishes. Cell surface markers were determined by a flow cytometric analysis, and the in vitro differentiation to chondrocytes, osteocytes or adipocytes was confirmed by staining with a specific dye. After four passages, these isolated cells displayed the typical morphology of mesenchymal cells in traditional 2-D cultures, and were grown to form spherical colonies in 3-D collagen cultures. Flow cytometric analysis revealed that the unsorted cells from all of seven patient samples showed robust expression of typical mesenchymal marker CD29, CD44, CD73, CD90 and CD166, and the absence of CD34, CD79a, CD105, CD271, SSEA-4, Stro-1 and HLA-DR. In differentiation assays, these cells were induced in vitro to chondrocytes, osteocytes or adipocytes. In conclusion, this preliminary study suggests the presence of MSCs in the “discarded” PD effluents. Further characterization of the phenotypes of these MSCs and evaluation of their therapeutic potential, particularly for the prevention of PD failure, are needed.