The Peculiar Facets of Nitric Oxide as a Cellular Messenger: From Disease-Associated Signaling to the Regulation of Brain Bioenergetics and Neurovascular Coupling

In this review, we address the regulatory and toxic role of ^·NO along several pathways, from the gut to the brain. Initially, we address the role on ^·NO in the regulation of mitochondrial respiration with emphasis on the possible contribution to Parkinson’s disease via mechanisms that involve its interaction with a major dopamine metabolite, DOPAC. In parallel with initial discoveries of the inhibition of mitochondrial respiration by ^·NO, it became clear the potential for toxic ^·NO-mediated mechanisms involving the production of more reactive species and the post-translational modification of mitochondrial proteins. Accordingly, we have proposed a novel mechanism potentially leading to dopaminergic cell death, providing evidence that NO synergistically interact with DOPAC in promoting cell death via mechanisms that involve GSH depletion. The modulatory role of NO will be then briefly discussed as a master regulator on brain energy metabolism. The energy metabolism in the brain is central to the understanding of brain function and disease. The core role of ^·NO in the regulation of brain metabolism and vascular responses is further substantiated by discussing its role as a mediator of neurovascular coupling, the increase in local microvessels blood flow in response to spatially restricted increase of neuronal activity. The many facets of NO as intracellular and intercellular messenger, conveying information associated with its spatial and temporal concentration dynamics, involve not only the discussion of its reactions and potential targets on a defined biological environment but also the regulation of its synthesis by the family of nitric oxide synthases. More recently, a novel pathway, out of control of NOS, has been the subject of a great deal of controversy, the nitrate:nitrite:NO pathway, adding new perspectives to ^·NO biology. Thus, finally, this novel pathway will be addressed in connection with nitrate consumption in the diet and the beneficial effects of protein nitration by reactive nitrogen species.

     

Inborn errors of complex II--unusual human mitochondrial diseases.

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed.     

Archaeal complex II: 'classical' and 'non-classical' succinate:quinone reductases with unusual features.

Reversible succinate dehydrogenase (SDH) activities have been ubiquitously detected in organisms from the three domains of life. They represent constituents either of respiratory complexes II in aerobes, or of fumarate dehydrogenase complexes in anaerobes. The present review gives a survey on archaeal succinate:quinone oxidoreductases (SQRs) analyzed so far. Though some of these could be studied in detail enzymologically and spectroscopically, the existence of others has been deduced only from published genome sequences. Interestingly, two groups of enzyme complexes can be distinguished in Archaea. One group resembles the properties of SDHs known from bacteria and mitochondria. The other represents a novel class with an unusual iron-sulfur cluster in subunit B and atypical sequence motifs in subunit C which may influence electron transport mechanisms and pathways. This novel class of SQRs is discussed in comparison to the so-called 'classical' complexes. A phylogenetic analysis is presented suggesting a co-evolution of the flavoprotein-binding subunit A and subunit B containing the three iron-sulfur clusters.     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Effect of lidocaine and pyromecaine on the size of an experimental myocardial infarct

The experiments on rats with a 3-day myocardial infarction caused by the left coronary artery ligation have shown that multiple lidocaine and pyromecaine injections according to a given scheme decrease the size of the necrosis area. Drug effects were not related to their action on the blood supply of the ischemic area.     

Reasons for disorders of fatty acid oxidation in isolated heart mitochondria during ischemia

The influence of malate and cytochrome c on fatty acid oxidation under control and ischemic conditions was investigated. In the medium without malate, cytochrome did not make fatty acid oxidation decreased during ischemia return to normal. Oxidation in the media containing malate and cytochrome did not differ from control only when it was measured after preliminary oxidation of endogenous substrates. The ratio of palmitoyl-CoA and palmitoyl carnitine to the respiration rates at state 3 was unchanged at 60 min ischemia. Apparently, no changes in carnitine acyltransferase playing a role in oxidation of palmitoyl-CoA took place. Thus, the decrease of fatty acid oxidation at early periods of ischemia is largely caused by a reduction in the content of cytochrome c and intermediates of Krebs cycle in the mitochondria.     

Purification and various properties of asialoglycoprotein receptors from the mouse liver

The receptor for asialoglycoproteins was isolated from murine liver and was purified by means of biospecific chromatography on sepharose-Asialo-orosomucoid. The obtained receptor with an absorption maximum at 277 nm binds to the nonreducing terminal galactosyl residues of glycoproteins similar to the receptors from liver of other mammalians. The interaction between this receptor and desialylated glycoproteins requires the presence of calcium. The dependence of specific binding on the concentration of [125I]acialo-orosomucoid used as a ligand gives a saturating curve. The dissociation constant for the receptor-ligand complex is 0.4 X 10(-9) M. Similar to asialo-orosomucoid, the receptor binds the p-aminophenyl-beta-D-galactopyranoside derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase synthesized by us earlier. Possible use of the asialoglycoprotein receptor as a highly specific carrier transporting the modified acid alpha-glucosidase to hepatocyte lysosomes is discussed.     

Relationship between postabsorptive respiratory exchange ratio and plasma free fatty acid concentrations

The relationship between overnight postabsorptive (fasting) respiratory exchange ratio (RER) and plasma FFA concentrations was addressed using data from three separate protocols, each of which involved careful control of the antecedent diet. Protocol 1 examined the relationship between fasting RER and the previous daytime RER. In Protocol 2 fasting, RER and plasma palmitate concentrations were measured in 29 women and 31 men (body mass index <30 kg·m⁻²). Protocol 3 analyzed data from Nielsen et al. (Nielsen, S., Z. K. Guo, J. B. Albu, S. Klein, P. C. O''Brien, M. D. Jensen. 2003. Energy expenditure, sex and endogenous fuel availability in humans. J. Clin. Invest. 111: 981-988.) to understand how fasting RER and palmitate concentrations relate within individuals during four consecutive measurements. The results were as follows: 1) Fasting RER was correlated (r = 0.74, P < 0.001) with the previous day''s average RER, and less so with RER variability. 2) Fasting RER was correlated (r = −0.39, P = 0.007) with fasting plasma palmitate concentrations. 3) The pattern of the RER/palmitate relationship was similar within individuals and between individuals; a negative slope was observed significantly more often than a positive slope (χ² test; P < 0.001). Our findings suggest that, despite a fixed food quotient, the slight departures from energy equilibrium in a controlled General Clinical Research Center environment can effect plasma FFA concentrations. We suggest that including indirect calorimetry as part of FFA metabolism studies may aid in data interpretation.