Lipid peroxide metabolism in chemical carcinogenesis]

Lecithin and kephalin content in the microsomes and mitochondria of the rat liver, and also the activity of enzymatic and nonenzymatic systems of the phospholipid peroxidation showed a sharp change following 3,4-benzpyrene injection. Carcinogenesis is accompanied by significant changes in the lipid peroxides content and in the activity of the enzyme utilizing lipoperoxides (glutathion peroxidase, glutathion reductase). Accumulation of lipid peroxides in the rat liver in carcinogenesis was connected with disturbed balance of the generating systems and detoxication of lipid peroxides in the tumour is attributed to the high activity of the protective enzymatic systems and serves as a reflection of the adaptation mechanisms directed to the maintenance of a high pool of proliferating cells in the tumour.     

Inhibition by [corrected] ursolic acid of [corrected] calcium-induced mitochondrial permeability transition and release of two proapoptotic proteins

The possible inhibition by [corrected] ursolic acid (UA) of [corrected] mitochondrial permeability transition (MPT) in mouse liver was investigated to identify the mechanisms underlying the hepatoprotective effect of UA. The effect of UA on liver MPT induced by Ca2+ was assessed by measuring changes in mitochondrial volume, mitochondrial membrane potential (MMP), release of matrix Ca2+, and transfer of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from the intermembrane space to the cytoplasm. The results showed that obvious mitochondrial swelling, loss of MMP, and release of matrix Ca2+ occurred after the addition of 50 microM Ca2+. However, preincubation with 20, 50 or 100 microg ml(-1) UA significantly blocked the above changes. Addition of 100 microg ml(-1) UA inhibited on mitochondrial swelling by 73.2% after 5 min, while the MMP dissipating and Ca2+ releasing were, respectively, suppressed by 59.3% and 54.1% after 3 min. In addition, Western blot analysis showed Cyt c and AIF transferred from mitochondrial pellet to the supernatant after the addition of 50 microM Ca2+, but the process was significantly inhibited by various concentrations of UA. The results suggest that the mechanisms underlying the hepatoprotection of UA may be related to its direct inhibitory action on MPT.     

The X and Y chromosomes assemble into H2A.Z-containing [corrected] facultative heterochromatin [corrected] following meiosis

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1beta and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.     

Micromolar intracellular hydrogen peroxide disrupts metabolism by damaging iron-sulfur enzymes

An Escherichia coli strain that cannot scavenge hydrogen peroxide has been used to identify the cell processes that are most sensitive to this oxidant. Low micromolar concentrations of H2O2 completely blocked the biosynthesis of leucine. The defect was tracked to the inactivation of isopropylmalate isomerase. This enzyme belongs to a family of [4Fe-4S] dehydratases that are notoriously sensitive to univalent oxidation, and experiments confirmed that other members were also inactivated. In vitro and in vivo analyses showed that H2O2 directly oxidized their solvent-exposed clusters in a Fenton-like reaction. The oxidized cluster then degraded to a catalytically inactive [3Fe-4S] form. Experiments indicated that H2O2 accepted two consecutive electrons during the oxidation event. As a consequence, hydroxyl radicals were not released; the polypeptide was undamaged; and the enzyme was competent for reactivation by repair processes. Strikingly, in scavenger-deficient mutants, the H2O2 that was generated as an adventitious by-product of metabolism (<1 microm) was sufficient to damage these [4Fe-4S] enzymes. This result demonstrates that aerobic organisms must synthesize H2O2 scavengers to avoid poisoning their own pathways. The extreme vulnerability of these enzymes may explain why many organisms, including mammals, deploy H2O2 to suppress microbial growth.     

Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.     

Contribution of the conserved amino acids of the melanocortin-4 receptor in [corrected] [Nle4,D-Phe7]-alpha-melanocyte-stimulating [corrected] hormone binding and signaling

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for alpha-melanocortin-stimulating hormone (alpha-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for alpha-MSH binding and signaling. alpha-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and alpha-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter alpha-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for alpha-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu(100) in TM2, Asp(122), Asp(126) in TM3 and Trp(258), Phe(261), His(264) in TM6 are involved in alpha-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for alpha-MSH binding and signaling.     

Cell-penetrating peptides: [corrected] from inception to application

Despite continuing advances in the development of macromolecules, including peptides, proteins, and oligonucleotides, for therapeutic purposes, the successful application of these hydrophilic molecules has so far been hampered by their inability to efficiently traverse the cellular plasma membrane. The discovery of a class of peptides (cell-penetrating peptides, CPPs) with the ability to mediate the non-invasive and efficient import of a whole host of cargoes, both in vitro and in vivo, has provided a new means by which the problem associated with cellular delivery can be circumvented. A complete understanding of the translocation mechanism(s) of CPPs has so far proven elusive. Initial studies indicated an ATP-independent, non-endocytotic mechanism, dependent on direct peptide-membrane interactions, making it an enticing challenge from a biophysical point of view. However, recent evidence cast doubt on many of the earlier results, and led to a re-evaluation of the translocation mechanism of CPPs. In this review a brief history of the field will be given, followed by an introduction to some of the better known and more widely used CPPs, including some of their current applications, and finally a discussion of the translocation mechanism(s) and the controversies surrounding it.     

Properties of an hydrogen peroxide resistant Proteus mirabilis mutant]

A peroxide-resistant mutant (PR) was isolated from Proteus mirabilis using the hydrogen peroxide mutagenic property. Under the same conditions, resistance of mutant PR bacteria to H2O2 was 50 to 100 times greater than that of the wild type. The total amount of catalase produced by P. mirabilis PR was on the average 10 times greater than that of the wild type. When PR bacteria were subjected to high doses of H2O2 (150mM), the determination of catalasic activity in vivo increased; paradoxically, there was a net decrease in the activity of the solubilized catalase after the breakdown of the cells. The hypothesis of an enzyme transfer from the inside towards the periphery of the cells is discussed. The behavior of a membrane enzyme (L-phenylalanine oxidase) of the PR mutant shows that H2O2 may cause lesions way up to the internal membrane of bacteria.     

Efficient secretion of Bacillus amyloliquefaciens alpha-amylase by [corrected] its own signal peptide from Saccharomyces cerevisiae host cells [corrected

The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm.     

AIDS and evidence: interrogating [corrected] some Ugandan myths

Uganda is invoked as a metaphor for a host of arguments and insights about HIV/AIDS. However, much of what has been asserted about the country is not based on the available evidence. This paper reviews findings by epidemiologists and anthropologists, and draws on the author's experiences of researching in the country since the early 1980s. It comments on various myths about HIV/AIDS in Uganda, including myths about the origin and dissemination of the disease, about the links between HIV/AIDS and war, and about declining rates of infection. It shows that much less is known about Uganda than is commonly supposed, and it offers some alternative hypotheses for interpreting HIV prevalence and incidence data. In particular it draws attention to the importance of mechanisms for social compliance. It concludes by raising concerns about the current enthusiasm for provision of anti-retroviral drugs.     

Elimination of senescent neutrophils by TNF-related apoptosis-inducing [corrected] ligand

Neutrophils are phagocytic effectors which are produced in the bone marrow and released into the circulation. Thereafter, they are either recruited to sites of inflammation or rapidly become senescent, return to the bone marrow, and undergo apoptosis. Stromal cell-derived factor 1 (SDF-1) coordinates the return of senescent neutrophils to the bone marrow by interacting with CXCR4 that is preferentially expressed on senescent neutrophils. We demonstrate that CXCR4 ligation by SDF-1 or other CXCR4 agonists significantly increases the expression of both TNF-related apoptosis-inducing [corrected] ligand (TRAIL) and of the death-inducing TRAIL receptors on neutrophils, which confers an acquired sensitivity to TRAIL-mediated death and results in TRAIL-dependent apoptosis. In vivo administration of TRAIL antagonists results in neutrophilic accumulation within the bone marrow and a reduction in neutrophil apoptosis; conversely recombinant TRAIL administration reduced neutrophil number within bone marrow. Thus, SDF-1 ligation of CXCR4 causes the parallel processes of chemotaxis and enhanced TRAIL and TRAIL death receptor expression, resulting in apoptosis of senescent neutrophils upon their return to the bone marrow.     

Isolation from poultry litter and characterization [corrected] of Staphylococcus spp. capable of growth in high phosphate conditions [corrected

AIMS: To isolate and characterize micro-organisms from poultry litter capable of growing under phosphate concentrations typical of poultry litter. METHODS AND RESULTS: Poultry litter extracts were plated onto brain-heart infusion medium (BHI) containing an additional 0.75 mol l(-1) phosphate (BHI-P). Colonies were screened for the presence of inclusion granules with five being selected for further study. All strains displayed identical biochemical characteristics consistent with Staphylococcus spp. and grouped with Staphylococcus spp. by comparative 16S rDNA analysis. Thus all five strains were identified as such. All strains displayed elevated intracellular phosphate levels when cultured in BHI-P broth (0.417-0.600 microg phosphate mg(-1) protein) vs BHI broth (0.075-0.093 microg phosphate mg(-1) protein). When grown using an austere semi-defined medium or BHI-P, Staph. sp. #7 displayed similar elevated intracellular phosphate levels compared with growth in BHI. CONCLUSIONS: Poultry litter contains novel Staphylococcus spp. capable of robust growth when exposed to phosphate levels comparable with that typically found in poultry litter. Data suggest intracellular phosphate levels in these strains increase in response to increasing phosphate in the medium or austere medium conditions. Intracellular phosphate did not reach levels comparable with known hyper-accumulating micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest poultry litter possesses a resident microflora that thrives and accumulates intracellular phosphate in response to high phosphate conditions.     

Inactivation of murine norovirus 1, coliphage phiX174, and Bacteroides [corrected] fragilis phage B40-8 on surfaces and fresh-cut iceberg lettuce by hydrogen peroxide and UV light

In this study, the inactivating properties of liquid hydrogen peroxide (L-H(2)O(2)), vaporized hydrogen peroxide (V-H(2)O(2)), UV light, and a combination of V-H(2)O(2) and UV light were tested on murine norovirus 1 (MNV-1) and bacteriophages (φX174 and B40-8) as models for human noroviruses. Disinfection of surfaces was examined on stainless steel discs based on European Standard EN 13697 (2001). For fresh-produce decontamination, a mixture of the viruses was inoculated onto shredded iceberg lettuce and treated after overnight incubation at 2°C. According to our results, L-H(2)O(2) (2.1%) was able to inactivate MNV-1 and φX174 on stainless steel discs by approximately 4 log(10) units within 10 min of exposure, whereas for B40-8, 15% of L-H(2)O(2) was needed to obtain a similar reduction in 10 min. Only a marginal reduction (≤1 log(10) unit after 5 min of exposure) by V-H(2)O(2) (2.52%) was achieved for the tested model viruses, although in combination with UV light, a 4-log(10)-unit decrease within 5 min of treatment was observed on stainless steel discs. Similar trends were observed for the decontamination of shredded iceberg lettuce, but the viral decline was reduced. These results demonstrated that both L-H(2)O(2) and a combination of V-H(2)O(2) and UV light can be used for norovirus inactivation on surfaces; V-H(2)O(2) (2.52%) in combination with UV light is promising for decontamination of fresh produce with much less consumption of water and disinfectant.     

Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.     

Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination.

The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.     

Role of catalase in metabolism of hydrogen peroxide by the perfused rat heart

Cardiac metabolism of H2O2 was studied by determining the concentration dependence for H2O2-stimulated release of GSSG, an indicator for flux through the glutathione peroxidase pathway, in perfused heart preparations. Treatment of rats with aminotriazole in vivo inhibited heart catalase by 83% and shifted the dose-response curve for GSSG release toward lower H2O2 concentrations. In aminotriazole-treated rats, 50 microM H2O2 elicited a maximal rate of GSSG release (about 5 nmol GSSG/min per g heart), whereas 200 microM H2O2 was necessary for obtaining a similar rate of GSSG release in control rat hearts. The results show that catalase, although present at low levels of activity in the heart compared to other organs, functions as a major route for detoxication of H2O2 in the myocardium.