Search

The signals initiated by skeletal muscle contraction and exercise involve several biochemical mechanisms both in the short- and long-time range that are under the control of thyroid hormones as genomic as well as nongenomic mechanisms. The effect of thyroid hormones on skeletal muscle has been studied both in cells in culture and in vivo. In L- 6 myoblasts thyroid hormone increases intracellular calcium and pH. Administration of a single injection of thyroid hormone in rat i.p. leads to a rapid rise in T3 about 50-fold, that peaked at 2 hours. The effect of this treatment was evaluated on signaling kinases, in particular on the phosphorylation of the p38MAPK pathway, and AMP-activated protein kinase. Both were activated and both can be a downstream target of the integrin αvβ3, the reported plasma membrane receptor for thyroid hormones. Contractile activity leads to mitochondrial biogenesis within skeletal muscle, which gives rise to signaling pathways leading to the expression of transcription factors. These are activated by a Ca2-dependent release, p38 and AMP-activated protein kinase. The activated transcription factors such as nuclear respiratory factor 1, interact with the coactivator PGC-1α to stimulate the expression of nuclear genes encoding mitochondrial proteins. This sequence of events can be induced not only by physical exercise, but also by thyroid hormone (T3) treatment in vivo, particularly in slow twitch skeletal muscle. In conclusion, the old statement that thyroid hormone helps to keep the steady state, is still valid, furthermore it contributes to a good performance of skeletal muscle as exercise does.

Antioxidant therapy is the novel frontier to prevent and treat an impressive series of severe human diseases, and the search for adequate antioxidant drugs is fervent. Cerium oxide nanoparticles (nanoceria) are redox-active owing to the coexistence of Ce3+and Ce4+oxidation states and to the fact that Ce3+defects, and the compensating oxygen vacancies, are more abundant at the surface. Nanoceria particles exert outstanding antioxidant effects in vivoacting as well-tolerated anti-age and anti-inflammatory agents, potentially being innovative therapeutic tools. However, the biological antioxidant mechanisms are still unclear. Here, the analysis on two leukocyte cell lines undergoing apoptosis viaredox-dependent or independent mechanisms revealed that the intracellular antioxidant effect is the direct cause of the anti-apoptotic and prosurvival effects of nanoceria. Doping with increasing concentrations of Sm3+, which progressively decreased Ce3+without affecting oxygen vacancies, blunted these effects, demonstrating that Ce3+/Ce4+redox reactions are responsible for the outstanding biological properties of nanoceria.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH,S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys47and Cys101residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys47lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys47/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (Ki< 10−12m) and triggers negative cooperativity in the vacant subunit (Ki= 10−9m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr7and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

No Abstract Supporting information for this article is available on the WWW under http://www.angewandte.com or from the author.

No Abstract Supporting information for this article is available on the WWW under http://www.angewandte.com or from the author.

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Gua-nídine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Kivalue of 10-6M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.

The intermediate CuI‐semiquinone radical species in the catalytic mechanism of copper‐amine oxidase from Lens esculentaand Pisum sativumseedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped‐flow and temperature‐jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Gua-nídine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Kivalue of 10-6M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.

Abstract:  The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k c value is different from that of native or Cu-fully-reconstituted enzyme, while K m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

AbstractIn this review, inhibitors of plant copper amine oxidases from Lens esculentaseedlings, Pisum sativumseedlings, and Euphorbia characiaslatex are described. Reversible competitive inhibitors and non-competitive inhibitors, irreversible active-site directed inhibitors and mechanism-based inactivators are reviewed in regard to their mechanisms of action.

Copper amine oxidase was shown to be able to oxidize kynuramine to the corresponding aldehyde, which spontaneously rearranged to 4‐hydroxyquinoline. Under anaerobic conditions, the native enzyme oxidized one equivalent of kynuramine and released one equivalent of aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry under anaerobic as well as aerobic conditions. These findings demonstrate unequivocally that copper‐free amine oxidase can oxidize substrates with a single incomplete turnover. © 1998 John Wiley & Sons, Ltd.

Experimental and clinical data suggest that oxygen and/or glucose deprivation alters electrical transmission in the brain and generates free radicals, which may mediate neuronal death. We have analyzed the effects of oxygen and/or glucose deprivation on both excitatory transmission, by measuring field potential amplitude, and free radical production, by using electron spin resonance (ESR) spectroscopy, in a corticostriatal slice preparation. Combined oxygen and/or glucose deprivation (ischemia) lasting 10 to 20 minutes induced a long-term depression of field potential amplitude. The ascorbyl radical could only be detected in brain slices during the reperfusion-phase after 30 minutes of ischemia. It appeared in the early minutes after the washout of ischemic medium and remained stable throughout the reperfusion phase. This radical was never detected in the external medium. Ischemia induced only a slight, but progressive, release of lactate dehydrogenase (LDH) into the external medium during the reperfusion phase. In contrast, exposure of slices to hypoxia or hypoglycemia alone resulted in transient depression of field potential amplitude, and no generation of ascorbyl radicals was observed on reperfusion. We propose that the long-lasting loss of electrical signals is the early sign of neuronal damage during ischemia. On the other hand, ascorbyl radical formation may be considered an indicator of neuronal injury after prolonged energy deprivation.

Recent work on the structure of the CuAsite of cytochrome c oxidase is described. Synthetic model systems containing two thiolate sulfur and two nitrogen donors are reviewed. The electronic model of CuAis discussed and a new dynamic model, which accounts for the atypical ESR properties of this enzyme, is suggested.

Protein‐radical enzymes use a free radical located on an intrinsic amino acid residue as a cofactor. The amino acid involved can be a tyrosine (ribonucleotide reductase, photosystem II, prostaglandin H synthase), a modified tyrosine (amine oxidase, galactose oxidase), a tryptophan (cytochrome cperoxidase), a modified tryptophan (methylamine dehydrogenase) or a glycine (ribonucleotide reduetase, pyruvate formate lyase). The mechanistic role of these radicals appears to be that of a one‐electron gate, allowing the separation of single reducing equivalents in time and space.

Copper amine oxidase was shown to be able to oxidize kynuramine to the corresponding aldehyde, which spontaneously rearranged to 4-hydroxyquinoline. Under anaerobic conditions, the native enzyme oxidized one equivalent of kynuramine and released one equivalent of aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry under anaerobic as well as aerobic conditions. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single incomplete turnover. © 1998 John Wiley & Sons, Ltd.

Protein-radical enzymes use a free radical located on an intrinsic amino acid residue as a cofactor. The amino acid involved can be a tyrosine (ribonucleotide reductase, photosystem II, prostaglandin H synthase), a modified tyrosine (amine oxidase, galactose oxidase), a tryptophan (cytochrome cperoxidase), a modified tryptophan (methylamine dehydrogenase) or a glycine (ribonucleotide reduetase, pyruvate formate lyase). The mechanistic role of these radicals appears to be that of a one-electron gate, allowing the separation of single reducing equivalents in time and space.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT‐IR absorption, CD, 2D‐NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14‐mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in‐depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical‐containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC‐labeled analogs in view of the poorly reactive α‐amino function of this very bulky residue and the specific requirements of its free‐radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α‐helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.