The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

The role of dicarbonyl compounds in non-enzymatic crosslinking: a structure-activity study

The Maillard reaction is a complex network of reactions that has been shown to result in the non-enzymatic crosslinking of proteins. Recent attention has focussed on the role of alpha-dicarbonyl compounds as important in vivo contributors to protein crosslinking but, despite extensive research, the molecular mechanisms of the crosslinking reaction remain open to conjecture. In particular, no relationship between the structure of the carbonyl-containing compounds and their activity as crosslinking agents has been established. In an effort to elucidate a structure-reactivity relationship, a wide range of dicarbonyl compounds, including linear, cyclic, di-aldehyde and di-ketone compounds, were reacted with the model protein ribonuclease A and their crosslinking activity assessed. Methylglyoxal and glutaraldehyde were found to be the most efficient crosslinkers, whilst closely related molecules effected crosslinking at a much lower rate. Cyclopentan-1,2-dione was also shown to be a reactive crosslinking agent. The efficiency of methylglyoxal and glutaraldehyde at crosslinking is thought to be related to their ability to form stable heterocyclic compounds that are the basis of protein crosslinks. The reasons for the striking reactivity of these two compounds, compared to closely related structures is explained by subtle balances between competing pathways in a complex reaction network.     

Overview of coenzyme A metabolism and its role in cellular toxicity

Coenzyme A (CoASH) has a clearly defined role as a cofactor for a number of oxidative and biosynthetic reactions in intermediary metabolism. Formation of acyl-CoA thioesters from organic carboxylic acids activates the acid for further biotransformation reactions and facilitates enzyme recognition. Xenobiotic carboxylic acids can also form CoA-thioesters, and the resulting acyl-CoA may contribute to the compound's toxicity. Generation of an unusual or poorly-metabolized acyl-CoA from a xenobiotic may lead to cellular metabolic dysfunction through several types of mechanisms including: (1) inhibition of key metabolic enzymes by the acyl-CoA; (2) sequestration of the total cellular CoA pool as the unusual acyl-CoA; (3) physical-chemical effects of the acyl-CoA; and (4) sequestration and depletion of carnitine as the acyl group is transformed from the acyl-CoA to form the corresponding acylcarnitine. Many of these toxicities are similar to sequelae observed in the inherited organic acidurias in which endogenously-generated acyl-CoAs accumulate secondary to an enzymopathy. Insights into the cellular mechanisms of xenobiotic acyl-CoA accumulation have been derived from model systems developed to understand organic acidemias, such as the methylmalonyl-CoA accumulation of the methylmalonic acidurias. The relevance of acyl-CoA accretion to human pathophysiology has now been well established, and identification of the relevant mechanism of toxicity can allow implementation of strategies to minimize the metabolic injury. Additionally, recognition of the potential for acyl-CoA mediated xenobiotic injury should result in improved rational drug design and earlier recognition of such toxicity when it develops.     

The non-enzymatic hydrolysis of oligoribonucleotides VI. The role of biogenic polyamines.

Single-stranded oligoribonucleotides containing UA and CA phosphodiester bonds can be hydrolyzed specifically under non-enzymatic conditions in the presence of spermidine, a biogenic amine found in a wide variety of organisms. In the present study, the rate of oligonucleotide and tRNA(i)(Met)hydrolysis was measured in the presence of spermidine and other biogenic amines. It was found that spermine [H(3)N(+)(CH(2))(3)(+)NH(2)(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] and putrescine [H(3)N(+)(CH(2))(4)(+)NH(3)] can replace spermidine [H(3)N(+)-(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] to induce the hydrolysis. For all three polyamines, a bell-shaped cleavage rate versus concentration relationship was observed. The maximum rate of hydrolysis was achieved at 0.1, 1.0 and 10 mM spermine, spermidine and putrescine, respectively. Moreover, we found that the hydrolysis requires at least two linked amino groups since two aminoalcohols, 2-aminoethanol and 3-aminopropanol, were not able to induce the cleavage of the phospho-diester bond. The optimal cleavage rate of the oligo-ribonucleotides was observed when amino groups were separated by tri- or tetramethylene linkers. The methylation of the amino groups reduced the ability of diamines to induce oligoribonucleotide hydrolysis. Non-enzymatic cleavage of tRNA(i)(Met)from Lupinus luteus and tRNA(i)(Met)from Escherichia coli demonstrate that both RNAs hydrolyze as expected from principles derived from oligoribonucleotide models.     

Organocatalysis with endogenous compounds: Towards novel non-enzymatic reactions

The aldol reaction of the endogeneous compounds acetone and methylglyoxal has been studied using organocatalysis in relation to biologically relevant non-enzymatic reactions. Under preparative conditions, 3-hydroxy-2,5-hexadione, known as Henze’s ketol, is formed in high yield and with enantioselectivities up to 88% ee. Furthermore, Henze’s ketol is also formed under simulated physiological conditions at micromolar scale, indicating that this reaction might take place in living organisms.     

Regulation of stearoyl-CoA desaturase genes: role in cellular metabolism and preadipocyte differentiation

The degree of fatty acid unsaturation in cell membrane lipids determines membrane fluidity, whose alteration has been implicated in a variety of disease states including diabetes, obesity, hypertension, cancer, and neurological and heart diseases. Stearoyl-CoA desaturase (SCD) is a key rate-limiting enzyme in the synthesis of unsaturated fatty acids by insertion of a cis-double bond in the Delta9 position of fatty acid substrates. Palmitate and stearate are the preferred substrates, which are converted to palmitoleate and oleate, respectively. These monounsaturated fatty acids are the major constituents of cellular membrane phospholipids and triacylglycerol stores found in adipose tissue. Two mouse and rat SCD genes (SCD1 and SCD2) have been cloned and characterized. During the differentiation of 3T3-L1 preadipocytes into adipocytes, SCD1 and SCD2 mRNAs are induced concomitant with increased de novo synthesis of palmitoleate and oleate. The physiological significance of expressing the two isoforms in the adipocytes is currently unknown. In this review we discuss the role of the SCD isoforms in metabolism and the recent findings on the differential regulation of mouse SCD genes by the antidiabetic thiazolidinediones (TZDs), during preadipocyte differentiation.     

The role of humoral and cellular mediators in enhanced mammary inflammatory reactions to staphylococcal infection in systemically immunized ewes

Prior systemic immunization with live Staphylococcus aureus vaccine enhances the early recruitment of neutrophils into nonlactating mammary glands infected with staphylococci. The study investigates the role of humoral and cellular mediators in this phenomenon. Intramammary infusion of bacteria suspended in immune sheep serum did not enhance the inflammatory response to infection in nonimmunized ewes despite the presence of complement in the infused serum. Infusion of complement activated by incubation with zymosan evoked a massive neutrophil influx into mammary secretions by 4 hr after infusion. Hemolytic complement activity was not detected in mammary secretions of immunized or nonimmunized ewes. These findings indicate that, despite the inflammatory effect of complement activation, humoral immune factors did not promote neutrophil migration into infected glands. Mammary glands of systemically immunized ewes stimulated 5 days previously with staphylococcal soluble antigens (SSA) supported larger neutrophil influxes during staphylococcal infection than contralateral glands stimulated with endotoxin 5 days prior to infection. Exudates of SSA-stimulated glands had significantly higher cell concentrations, prior to infection, than endotoxin-stimulated glands; however elevated cell concentrations in endotoxin-stimulated glands of nonimmune ewes did not support enhanced inflammatory responses. These findings suggest that qualitative but not quantitative characteristics of mammary leucocytes influence the inflammatory response to infection in systemically immunized ewes.     

Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism

Most mammalian cells receive exogenous folate from the bloodstream in the form of 5-methyltetrahydropteroylmonoglutamate (CH3-H4PteGlu1). Because this folate derivative is a very poor substrate for folylpolyglutamate synthetase, the enzyme that adds glutamyl residues to intracellular folates, CH3-H4PteGlu1 must first be converted to tetrahydropteroylmonoglutamate (H4PteGlu1), 10-formyltetrahydropteroylmonoglutamate (CHO-H4PteGlu1), or dihydrofolate (H2folate), which are excellent substrates for folylpolyglutamate synthetase. Polyglutamylation is required both for retention of intracellular folates and for efficacy of folates as substrates for most folate-dependent enzymes. Two enzymes are known that will react with CH3-H4PteGlu1 in vitro, methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyltransferase (cobalamin-dependent methionine synthase). These studies were performed to assess the possibility that methylenetetrahydrofolate reductase might catalyze the conversion of CH3-H4PteGlu1 to CH2-H4PteGlu1. CH2-H4PteGlu1 is readily converted to CHO-H4PteGlu1 by the action of methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, and these enzyme activities show very little preference for folypolyglutamate substrates as compared with folylmonoglutamates. We conclude from in vitro studies of the enzyme that methylenetetrahydrofolate reductase cannot convert CH3-H4PteGlu1 to CH2-H4PteGlu1 under physiological conditions and that uptake and retention of folate will be dependent on methionine synthase activity.