Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats

Abstract

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p?≤?0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p?=?0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.     

Effects of Parenteral Lipid Infusion on DNA Damage in Very Low Birth Weight Infants

Very low birth weight (VLBW) infants are known to have poorly developed antioxidant system and may be at increased risk for radical damage. Previous studies have reported higher levels of lipid peroxide products in lipid emulsion used for parenteral nutrition. To examine the direct effects of parenteral lipid infusion on DNA damage in VLBW infants, we measured urinary 8-hydroxydeoxyguanosine (8-OHdG) levels in VLBW infants before, during, and after the parenteral lipid infusion. In both the lipid-infused and lipid-free groups, urinary 8-OHdG excretion levels at 14 days old were significantly ( p <0.01) lower than those at 2 and 7 days old. However, there were no significant differences in urinary 8-OHdG excretion levels between the lipid-infused and lipid-free groups at 2, 7, and 14 days old. Our results suggest that parenteral lipid infusion does not cause oxidative DNA damage, but irrespective of the infusion DNA damage during the first week of life is enhanced when compared with 14 days after birth in VLBW infants.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

A novel peptide specifically targeting ovarian cancer identified by in vivo phage display

Discovery of peptide ligands that can target human ovarian cancer and deliver chemotherapeutics offers new opportunity for cancer therapy. The advent of phage‐displayed peptide library facilitated the screening of such peptides. In vivo screening that set in a microanatomic and functional context was applied in our study, and a novel peptide WSGPGVWGASVK targeting ovarian cancer was isolated. The phage clone PC3‐1 displaying peptide WSGPGVWGASVK can gain effective access to accumulate in the tumor sites after intravenous injection while reducing its accumulation in normal organs. Positive immunostaining of PC3‐1 was located in both sites of tumor cells and tumor blood vessels, which resulted in a diffuse binding pattern through the tumor. In vitro study results confirmed the capability of peptide WSGPGVWGASVK binding to and being internalized by both tumor cells and angiogenic endothelial cells. Flow cytometry analysis revealed that the peptide bound to SKOV3 cells with Kd value of 5.43 ± 0.4 μM. Taken together, it suggested that peptide WSGPGVWGASVK is a lead candidate for delivering therapeutics to penetrate into tumors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Hydrocaffeic and p-coumaric acids,natural phenolic compounds,inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo

This paper studied the effect on UV-B ocular damage of 10µm hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5µm HCAF+5µm p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m²) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m²). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E₂ (PGE₂) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.     

The bile acid membrane receptor TGR5: a novel pharmacological target in metabolic,inflammatory and neoplastic disorders

Abstract

TGR5 is the G-protein–coupled bile acid-activated receptor, found in many human and animal tissues. Considering different endocrine and paracrine functions of bile acids, the current review focuses on the role of TGR5 as a novel pharmacological target in the metabolic syndrome and related disorders, such as diabetes, obesity, atherosclerosis, liver diseases and cancer. TGR5 ligands improve insulin sensitivity and glucose homeostasis through the secretion of incretins. The bile acid/TGR5/cAMP signaling pathway increases energy expenditure in brown adipose tissue and skeletal muscle. Activation of TGR5 in macrophages inhibits production of proinflammatory cytokines and attenuates the development of atherosclerosis. This receptor has been detected in many cell types of the liver where it has anti-inflammatory effects, thus reducing liver steatosis and damage. TGR5 also modulates hepatic microcirculation and fluid secretion in the biliary tree. In cell culture models TGR5 has been linked to signaling pathways involved in metabolism, cell survival, proliferation and apoptosis, which suggest a possible role of TGR5 in cancer development. Despite the fact that TGR5 ligands may represent novel drugs for prevention and treatment of different aspects of the metabolic syndrome, clinical studies are awaited with the perspective that they will complete TGR5 biology and identify efficient and safe TGR5 agonists.     

Purification and Characterization op a 31-Kilodalton Iron-Regulated Periplasmic Protein from Pasteurella Haenolytica A1

ABSTRACT

A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9, The secondary structure of the protein was determined by circular dichroism and contained 16% α-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

Comparison of insecticidal paint and deltamethrin against Triatoma infestans (Hemiptera: Reduviidae) feeding and mortality in simulated natural conditions

The vector of Chagas disease, Triatoma infestans, is largely controlled by the household application of pyrethroid insecticides. Because effective, large‐scale insecticide application is costly and necessitates numerous trained personnel, alternative control techniques are badly needed. We compared the residual effect of organophosphate‐based insecticidal paint (Inesfly 5A IGR? (I5A)) to standard deltamethrin, and a negative control, against T. infestans in a simulated natural environment. We evaluated mortality, knockdown, and ability to take a blood meal among 5^th instar nymphs. I5A paint caused significantly greater mortality at time points up to nine months compared to deltamethrin (Fisher's Exact Test, p < 0.01 in all instances). A year following application, mortality among nymphs in the I5A was similar to those in the deltamethrin (χ2 = 0.76, df=1, p < 0.76). At months 0 and 1 after application, fewer nymphs exposed to deltamethrin took a blood meal compared to insects exposed to paint (Fisher's Exact Tests, p < 0.01 and p < 0.01, respectively). Insecticidal paint may provide an easily‐applied means of protection against vectors of Chagas disease.     

Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3‐like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.