Redox metabolism in malaria: from genes,through biochemistry and pathology,to drugs

Abstract

The role of Saccharomyces cerevisiae flavohemoglobin (Yhb1) is controversial and far from understood. This study compares the effects of nitrosative and oxidative challenge on the yeast mutant lacking the YHB1 gene. Growth of the mutant was impaired by nitrosoglutathione and peroxynitrite, whereas increased sensitivity to reactive oxygen species was not observed. Increased levels of intracellular NO^? after incubation with NO^? donors were found in the mutants cells as compared to the wild-type cells. Deletion of the YHB1 gene was found to augment the reduction of Fe³⁺ by yeast cells which suggests that flavohemoglobin participates in regulation of the activity of plasma membrane ferric reductase(s).     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Level of nitrated proteins in the plasma,platelets and liver of patients with liver cirrhosis

Abstract

Over-expression of nitric oxide synthase (NOS) and nitric oxide (NO) formation are associated with the pathogenesis of liver cirrhosis. NO-related stress alters the functions of biomolecules, especially proteins, probably as a result of nitration. The aim of this study was to assess the level of protein nitration and its correlation with the severity of the disease. Liver cirrhosis patients with different grades of severity (grades A, B, and C according to the Child–Pugh classification) were enrolled in this study. Nitroprotein content, arginine, citrulline, NO in terms of total nitrite, nitrosothiol (RSNO) and protein carbonyls were measured in blood. Immunohistochemical detection of nitroprotein was carried out in liver sections of cirrhosis patients. A significant elevation in the levels of serum and platelet arginine, arginase, citrulline, plasma, and platelet nitroproteins, RSNO, total nitrite, protein carbonyls and also a significant amount of nitrated proteins by immunohistochemical detection in tissue were observed in cirrhosis patients. The alterations were highly significant in grade C patients with bleeding complications when compared to those of grade B and A patients. In platelets, both cytosolic and cytoskeletal proteins were found to be nitrated significantly. The level of nitrite seems to have positive correlation with the level of nitroproteins in different grades of cirrhosis. The level of nitroproteins in plasma, platelets and liver tissue can be correlated with the severity of liver cirrhosis.     

Role of nitric oxide in pathological responses of the lung to exposure to environmental/occupational agents

Abstract

Conflicting evidence exists as to whether nitric oxide expresses damaging/inflammatory or antioxidant/anti-inflammatory properties. Data presented in this review indicate that in vitro or in vivo exposure to selected environmental or occupational agents, such as asbestos, silica, ozone or lipopolysaccharide, can result in up-regulation of inducible nitric oxide synthase by alveolar macrophages and pulmonary epithelial cells. In the case of silica exposure, evidence consistently supports a damaging/inflammatory role of nitric oxide and/or peroxynitrite in the pathogenesis of lung disease. Although conflicting data have been reported, the majority of published studies suggest that nitric oxide plays a damaging role in pulmonary injury resulting from exposure to ozone or asbestos. In contrast, most information supports an anti-inflammatory role of nitric oxide following exposure to lipopolysaccharide. Further investigation is required to elucidate fully the mechanisms involved in determining the role of nitric oxide in the initiation and progression of various pulmonary diseases.     

Glucose autoxidation induces functional damage to proteins via modification of critical arginine residues

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.     

Maillard reaction products in tissue proteins: New products and new perspectives

Summary. The chemical modification of protein by nonenzymatic browning or Maillard reactions increases with age and in disease. Maillard products are formed by reactions of both carbohydrate- and lipid-derived intermediates with proteins, leading to formation of advanced glycation and lipoxidation end-products (AGE/ALEs). These modifications and other oxidative modifications of amino acids increase together in proteins and are indicators of tissue aging and pathology. In this review, we describe the major pathways and characteristic products of chemical modification of proteins by carbohydrates and lipids during the Maillard reactions and identify major intersections between these pathways. We also describe a new class of intracellular sulfhydryl modifications, Cys-AGE/ALEs, that may play an important role in regulatory biology and represent a primitive link between nonenzymatic and enzymatic chemistry in biological systems.     

In vivo magnetic resonance imaging tracking of SPIO-labeled human umbilical cord mesenchymal stem cells

Human umbilical cord mesenchymal stem cells (hUC-MSCs) can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticles, which produces low signal intensity on magnetic resonance imaging (MRI) in vitro. This study was to evaluate the feasibility of in vivo tracking for hUC-MSCs labeled by SPIO with noninvasive MRI. SPIO was added to cultures at concentrations equivalent to 0, 7, 14, 28, and 56 μg Fe/ml (diluted with DMEM/F12) and incubated for 16 h. Prussian Blue staining was used to determinate the labeling efficiency. Rats were randomly divided into three groups, control group, hUC-MSCs group, and SPIO-labeled hUC-MSCs group. All groups were subjected to spinal cord injury (SCI) by weight drop device. Rats were examined for neurological function. In vivo MRI was used to track SPIO-labeled hUC-MSCs transplanted in rats spinal cord. Survival and migration of hUC-MSCs were also explored using immunofluorescence. Significant improvements in locomotion were observed in the hUC-MSCs groups. There was statistical significance compared with control group. In vivo MRI 1 and 3 weeks after injection showed a large reduction in signal intensity in the region transplanted with SPIO-labeled hUC-MSCs. The images from unlabeled hUC-MSCs showed a smaller reduction in signal intensity. Transplanted hUC-MSCs engrafted within the injured rats spinal cord and survived for at least 8 weeks. In conclusion, hUC-MSCs can survive and migrate in the host spinal cord after transplantation, which promote functional recovery after SCI. Noninvasive imaging of transplanted SPIO-labeled hUC-MSCs is feasible.     

Role of globin moiety in the autoxidation reaction of oxymyoglobin: effect of 8 M urea.

It is in the ferrous form that myoglobin or hemoglobin can bind molecular oxygen reversibly and carry out its function. To understand the possible role of the globin moiety in stabilizing the FeO2 bond in these proteins, we examined the autoxidation rate of bovine heart oxymyoglobin (MbO2) to its ferric met-form (metMb) in the presence of 8 M urea at 25 degrees C and found that the rate was markedly enhanced above the normal autoxidation in buffer alone over the whole range of pH 5-13. Taking into account the concomitant process of unfolding of the protein in 8 M urea, we then formulated a kinetic procedure to estimate the autoxidation rate of the unfolded form of MbO2 that might appear transiently in the possible pathway of denaturation. As a result, the fully denatured MbO2 was disclosed to be extremely susceptible to autoxidation with an almost constant rate over a wide range of pH 5-11. At pH 8.5, for instance, its rate was nearly 1000 times higher than the corresponding value of native MbO2. These findings lead us to conclude that the unfolding of the globin moiety allows much easier attack of the solvent water molecule or hydroxyl ion on the FeO2 center and causes a very rapid formation of the ferric met-species by the nucleophilic displacement mechanism. In the molecular evolution from simple ferrous complexes to myoglobin and hemoglobin molecules, therefore, the protein matrix can be depicted as a breakwater of the FeO2 bonding against protic, aqueous solvents.     

Inhibition of inducible nitric oxide synthase prevents lipid peroxidation in osteoarthritic chondrocytes

Nitric oxide (NO) and the lipid peroxidation (LPO) product 4-hydroxynonenal (HNE) are considered to be key mediators of cartilage destruction in osteoarthritis (OA). NO is also known to be an important intermediary in LPO initiation through peroxynitrite formation. The aim of the present study was to assess the ability of the inducible NO synthase (iNOS) inhibitor N-iminoethyl-L-lysine (L-NIL) to prevent HNE generation via NO suppression in human OA chondrocytes and cartilage explants. Human OA chondrocytes and cartilage explants were treated with L-NIL and thereafter with or without interleukin-1beta (IL-1β) or HNE at cytotoxic or non-cytotoxic concentrations. Parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated. L-NIL stifled IL-1β-induced NO release, iNOS activity, nitrated proteins, and HNE generation in a dose-dependent manner. It also blocked IL-1β-induced inactivation of the HNE-metabolizing glutathione-s-transferase (GST). L-NIL restored both HNE and GSTA4-4 levels in OA cartilage explants. Interestingly, it also abolished IL-1β-evoked reactive oxygen species (ROS) generation and p47 NADPH oxidase activation. Furthermore, L-NIL significantly attenuated cell death and markers of apoptosis elicited by exposure to a cytotoxic dose of HNE as well as the release of prostaglandin E(2) and metalloproteinase-13 induced by a non-cytotoxic dose of HNE. Altogether, our findings support a beneficial effect of L-NIL in OA by (i) preventing the LPO process and ROS production via NO-dependent and/or independent mechanisms and (ii) attenuating HNE-induced cell death and different mediators of cartilage damage.     

In vivo cross-linking of proteins to mRNA in human cells

Human KB cells were irradiated with ultraviolet light to cross-link mRNA to its associated proteins. More than 75% of both the poly(A)-containing and the poly(A)-lacking mRNAs were cross-linked to proteins after 3 min irradiation. Glycerol gradient analysis showed that no significant RNA chain breakage occurred during this treatment. Cross-linked poly(A)-containing mRNA-protein complexes were purified by oligo(dT)cellulose chromatography in the presence of sodium dodecylsulphate. CsCl gradient analysis revealed that the low salt eluted particles had a buoyant density of about 1.47 g/cm³. To determine which proteins were cross-linked to mRNA, covalent mRNA-protein complexes, labeled in their RNA moiety, were exhaustively treated with nucleases. Polyacrylamide gel analysis showed that most of the residual RNA-radioactivity was covalently bound to proteins of 73000, 69000 and 52000 molecular weight.     

Targeting of tail-anchored proteins to yeast mitochondria in vivo.

Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.     

The role of SMC proteins in the responses to DNA damage

The SMC proteins form the cores of three protein complexes in eukaryotes, cohesin, condensin and the Smc5-6 complex. Cohesin holds sister chromatids together after DNA replication and is involved in both the repair of double-strand breaks by homologous recombination and the intra-S-phase checkpoint. Condensin assists in the condensation of chromosomes at mitosis and also has a role in checkpoint control pathways. The Smc5-6 complex is involved in a variety of DNA repair and damage response pathways by as yet unknown mechanisms, but is also associated with repair by homologous recombination.     

Role of binding proteins to IRS-1 in insulin signalling

Insulin elicits its divergent metabolic and mitogenic effects by binding to its specific receptor, which belongs to the family of receptor tyrosine kinases. The activated insulin receptor phosphorylates the intracellular substrate IRS-1, which then binds various signalling molecules that contain SRC homology 2 domains, thereby propagating the insulin signal. Among these IRS-1-binding proteins, the Grb2-Sos complex and the protein tyrosine phosphatase SHP-2 transmit mitogenic signals through the activation of Ras, and phosphoinositide 3-kinase is implicated in the major metabolic actions of insulin. Although substantial evidence indicates the importance of IRS-1 in insulin signal transduction, the generation of IRS-1-deficient mice has revealed the existence of redundant signalling pathways.     

Metabolism of 3-deoxyglucosone,an intermediate compound in the Maillard reaction,administered orally or intravenously to rats

Tha Amadori rearrangement compound, the product in the early step of the Maillard reaction of proteins with glucose, is known to be degraded into 3-deoxyglucosone (3DG), a 2-oxoaldehyde. In order to elucidate the metabolic pathway of 3DG, [¹⁴C]3DG was synthesized from [¹⁴C]-glucose and administered to rats orally and intravenously. 2 h after oral administration of [¹⁴C]3DG, the percentages of radioactivity (RaI%) in stomach, small intestine and urine were 3.9, 60 and 6.4%, respectively, while RaI% in liver, kidney, spleen, blood and CO₂ were less than 0.5%. The absorption rate of 3DG was obviously lower in comparison with that of glucose. 3 h after intravenous administration of [¹⁴C]3DG, the RaI% in urine was 72% and those in liver, kidney, spleen, blood and CO₂ were less than 1%. It therefore appeared that the absorbed 3DG was not biologically utilized by the rats, but was rapidly excreted in the urine. Some metabolites of [¹⁴C]3DG were detected in urine by TLC-autoradiography. The main metabolite was purified and identified as 3-deoxyfructose by FD-MS and ¹³C-NMR spectroscopy, indicating that the aldehyde group of 3DG was reduced to an alcohol.     

Nonenzymatic glycosylation of and oxidative damage to actin in vitro and in vivo

A study was made the influence exerted by non-enzymatic glycosylation (glycation) and oxidative destruction on structural and functional parameters of actin (free NH2-groups, advanced glycation end product and bityrosine cross-linking content, DNase inhibition by G-actin and myosin Mg(2+)-ATPase activation by F-actin). The functional properties of actin were shown to change under high molecular weight product formation and oxidative destruction: the extent of DNAase I inhibition decreases (from 70 to 40%) and the extent of myosin Mg(2+)-ATPase decreases (by 40%). Carnosine prevents actin oligomer formation and oxidative destruction which favours preservation of the protein functional properties.