Oxidation Of Biomolecules Research Articles

Anti-Microbial Effect of Different Time's Exposure of Ozonized Gas and Ozonized Water on Periodontal Pathogens : In Vitro Study

Background: the oral cavity is consider to be an open ecosystem, with the balance between the microorganism’s entrance and the defenses of the host. The initiation of periodontitis has been associated with restricted kinds of anaerobic bacteria, such as Aggregatibacter actinomycetemcomitans (A.a) and Porphyromonas gingivalis (P.g) in plaque subgingivally. Ozone has a biological effects on bacteria due to oxidation of bio-molecules and its toxins. The aim is to determine and compare the antimicrobial effect of gaseous ozone and ozonized water on the growth of isolated anaerobic bacteria (A.a and P.g) when exposed to different time intervals. Materials and methods:This experiment is done byozone generator OLYMPIC- III(600mg/hr) to generator the gaseous ozone (218ppm/W-air)which bypassed around the agar plates containing on of the isolated bacteria with different time intervals (1-10 minutes).And with special aeration stone for generation of ozonized water (0.6 ppm) with different time intervals (1-15 minutes). Results: Gaseousozone have a significant reduction in the bacterial growth on the agar plates for (A.a) was 7 minutes and (P.g) was 4 minutes. While ozonated water have also a significant reduction in the bacterial growth on the agar plates for (A.a) was 5 minutes and (P.g) was 4 minutes. Conclusion: Bothgaseousozone and ozonized water are a powerful antimicrobialeffects on anaerobic microorganism isolated from chronic periodontitis patients.

Correlation between serum free iron, glycated hemoglobin and insulin resistance in uncontrolled type-2 diabetic patients

Background: The link between iron and diabetes was first recognized in pathologic conditions—hereditary hemochromatosis and thalassemia—but high levels of dietary iron also impart diabetes risk. Free iron may contribute to the pathogenesis and progress of this disease and its complication. Iron causes hyperinsulinemia by decreasing the insulin uptake and metabolism by hepatocytes. Elevated iron stores are commonly found in insulin resistance. Iron in its free form is known to induce oxidation of biomolecules by producing harmful hydroxyl radicals. Aims and Objectives: In this study, we aimed to estimate and compare the serum levels of free iron in diabetes and healthy individuals. Methods and Materials: This study included 253 subjects in 2 groups. Group-I comprised 207 subjects with diabetes mellitus and Group-II comprised 46 healthy subjects. Blood sugar, free iron, and glycated hemoglobin were analyzed in blood samples using standard kits. The results of all the parameters were expressed as mean ± standard deviation. Student t-test was done to assess the statistical significance between 2 groups. The association between the parameters was studied by Pearson correlation. Result: In this study, we found a significant increase in serum free iron in Group-I (P < 0.01), when compared with Group-II. A significant correlation between the serum free iron and glycated hemoglobin (r = 0.59; P < 0.001) and fasting blood sugar (r = 0.43; P < 0.001). Conclusion: The elevated serum free iron in uncontrolled diabetes may contribute to oxidative stress which may be associated with complications of diabetes.

К вопросу о возможности аллогенной плацентарной терапии постовариоэктомических проявлений у больных, отягощенных гинекологическим раком, после противоопухолевого лечения

The results of a clinical-psychological prospective examination of 131 patients with cervical cancer, the uterus body and ovaries of stage I-III according to FIGO, reproductive age in total hysterovectomy and radiation suppression of ovaries isolated in three clinical groups are presented: group I - patients after combined and complex special treatment; Group II - patients after combined radiation treatment and group III - patients after combined and complex treatment, therapy of postovariochemical manifestations of which was carried out by traditional therapy. Identification of personal resources of stress-resistance was carried out using the copy-test of R.Lazarus. The intensity of oxidative processes in blood plasma was judged by the content of their carbonyl derivatives, revealed in the reaction with 2,4-dinitrophenylhydrazine. Results and discussion. For patients in the state of total hysterovarioectomy (groups I and III), the hyperactivity of the strategy Confrontation (76.8% and 76.4%, respectively) and Positive revaluation (67.8% and 72.6%) are a common phenomenon. Constructive strategies; Distant (73.8%) and Acceptance of responsibility (82.5%) are typical for patients of the II group who are in drug and radiation suppression. In the blood of all patients, increased oxidative potential was determined. Significant changes were demonstrated in the analysis of the intensity of induced (metal-catalyzed) OMB: at λ 370 nm and λ 530 nm in group I and group III (p<0.01): the level of oxidation products was significantly lower in the group II patients after SLT. Changes were demonstrated in the analysis of the intensity of induced (metal-catalyzed) OMB. The level of carbonyl groups was significantly lower in the group of patients II after SLT (p<0.01). After the therapy with allogeneic hydrolyzate of the placenta (AHP) with Melsmon of the APEC for 14 weeks, an objective orientation toward a decrease in the oxidative potential in the blood of patients was manifested. The serum level of the end products of OMB had an active decrease in comparison with the similar values in group I at λ 370 nm (1.3 times) and λ 430 nm (1.5 times); p<0.01. At λ 530 nm - groups I decrease by 1.4 times and II decrease by 1.6 times, p<0.01. The level of OMB in induced oxidation demonstrated a pronounced active decrease, especially at λ 530 (p<0.01). The study of induced AMB in the serum of Group III patients with gynecological cancer, the treatment of which was carried out by traditional methods in the PES did not show any statistical differences. In group I patients, the activity of non-constructive strategies is significantly reduced: confrontation (from 76.8% to 54.2%); flight/avoidance (from 64.2% to 38.6%). More actively, patients began to apply resources to constructive distancing strategies (activity increased from 42.4% to 68.3%); planning of problem solving (from 42.8% to 72.4%). The active decrease in the level of the carbonyl products of protein oxidation correlated with the improvement and stabilization of the psychological status in the form of non-constructive coping strategies Confrontation, Escape-avoidance in patients of group I (r=0.379, r=0.437, p<0.001) and group II (r=0.439, r=0.376, p<0.001). Conclusions. 1. In gynecological cancer cases examined in the PES state , there is an intensification of the processes of free radical oxidation of biomolecules. However, in patients with preserved ovaries, the oxidative processes are somewhat reduced. 2. Decrease in the levels of carbonyl oxidation products against the background of Melsmon therapy in patients with gynecological cancer in the state of PES testifies to the possibility of participation of the drug components in antiradical protection. 3. Evaluation of the results of sonographic monitoring of the abdominal and pelvic organs in patients included in the study did not reveal a worsening of the prognosis of the underlying disease against the background of allogeneic placental therapy. 4. Allogeneic placental therapy with Melsmon helps to improve the psychological status of gynecological cancer patients in the form of stress-overcoming.

Healing effect of Dillenia indica fruit extracts standardized to betulinic acid on ultraviolet radiation-induced psoriasis-like wounds in rats

Context: Dillenia indica Linn. (Dilleniaceae) is traditionally used to treat skin inflammation.Objective: This study evaluated the healing effect of Dillenia indica fruit extracts on induced psoriasis-like wounds in Wistar rats.Materials and methods: Extracts were standardized to betulinic acid, including an aqueous ethanolic extract (AEE), ethyl acetate extract (EAE) and petroleum ether extract. Effects against lipid peroxidation were assessed in vitro. Wounds were created at rat tails (n = 12). Topical treatments were applied once daily for 7 days (1 mL of AEE or EAE at 5 or 50 mg/mL). Maximal dose was defined by the extract solubility. A 10-fold lower dose was also tested. Positive and negative controls were treated with clobetasol (0.5 mg/mL) or excipient. Half of each group was euthanized for histology. The remaining animals were observed for 20 days for wound measurements.Results: Yields of AEE and EAE were 4.3 and 0.7%, respectively. Betulinic acid concentrations in AEE and EAE were 4.6 and 107.6 mg/g. Extracts neutralized lipid peroxidation in vitro at 0.02 μg/mL, accelerating healing at 50 mg/mL. Complete healing in mice treated with AEE occurred 16 days after wound induction. This time was 14 and 12 days in mice treated with EAE and clobetasol. Compared to orthokeratosis, parakeratosis was reduced by AEE (25%), EAE (45%) and clobetasol (55%). EAE caused superior protection against biomolecules oxidation of skin compared to AEE.Discussion and conclusion: EAE exhibited activity closer to that of clobetasol. Betulinic acid may be an active constituent, which should be assessed in future studies.

Protective activity of Hertia cheirifolia extracts against DNA damage, lipid peroxidation and protein oxidation

Context: Hertia cheirifolia L. (Asteraceae), a perennial shrub widely distributed in Northern Africa, is traditionally used to treat inflammatory disorders. Objective: The protective effect of methanol (Met E) and aqueous (Aq E) extracts of Hertia cheirifolia against DNA, lipid and protein oxidation was investigated. Materials and methods: Different concentrations (50–1000 μg/mL) of Hertia cheirifolia aerial part extracts were examined against DNA, lipid and protein oxidation induced by H2O2 + UV, FeSO4, and Fe3+/H2O2-ascorbic acid, respectively. The DPPH•, metal ion chelating, reducing power and β-carotene bleaching tests were conducted. Results: Both extracts were rich in polyphenols, flavonoids and tannins, and were able to scavenge DPPH• with IC50 values of 138 and 197 μg/mL, respectively. At 300 μg/mL, Aq E exerted stronger chelating effect (99%) than Met E (69%). However, Met E reducing power (IC50 = 61 μg/mL) was more than that of Aq E (IC50 = 193 μg/mL). Both extracts protected from β-carotene bleaching by 74% and 94%, respectively, and inhibited linoleic acid peroxidation. The inhibitory activity of Aq E extract (64%) was twice more than that of Met E (32%). Interestingly, both extracts protected DNA against the cleavage by about 96–98%. At 1 mg/mL, Met E and Aq E restored protein band intensity by 94–99%. Conclusions: Hertia cheirifolia exhibits potent antioxidant activity and protects biomolecules against oxidative damage; hence, it may serve as potential source of natural antioxidant for pharmaceutical applications and food preservation. This is the first report on the protective activity of this plant against biomolecule oxidation.

The effects of photodynamic treatment with new methylene blue N on the Candida albicans proteome.

Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface.

Novel bis-arylalkylamines as myeloperoxidase inhibitors: Design, synthesis, and structure-activity relationship study

Human myeloperoxidase (MPO) plays an important role in innate immunity but also aggravates tissue damage by oxidation of biomolecules at sites of inflammation. As a result from a recent high-throughput virtual screening approach for MPO inhibitors, bis-2,2′-[(dihydro-1,3(2H,4H) pyrimidinediyl)bis(methylene)]phenol was detected as a promising lead compound for inhibition of the MPO-typical two-electron oxidation of chloride to hypochlorous acid (IC50 = 0.5 μM). In the present pharmacomodulation study, 37 derivatives of this lead compound were designed and synthesized driven by comprehensive docking studies and the impact on the chlorination activity of MPO. We describe the structural requirements for optimum (i) binding to the heme periphery and (ii) inhibition capacity. Finally, the best three inhibitors (bis-arylalkylamine derivatives) were probed for interaction with the MPO redox intermediates Compound I and Compound II. Determined apparent bimolecular rate constants together with determination of reduction potential and nucleophilicity of the selected compounds allowed us to propose a mechanism of inhibition. The best inhibitor was found to promote the accumulation of inactive form of MPO-Compound II and has IC50 = 54 nM, demonstrating the successful approach of the drug design. Due to the similarity of ligand interactions between MPO and serotonine transporter, the selectivity of this inhibitor was also tested on the serotonin transporter providing a selectivity index of 14 (KiSERT/IC50MPO).

Medicinal attributes of major phenylpropanoids present in cinnamon

BackgroundExcessive production of free radicals has been implicated in many diseases including cancer. They are highly reactive and bring about oxidation of biomolecules i.e., proteins, lipids and nucleic acids which are associated with many degenerative diseases. Natural products acting as antioxidants have ability to neutralize free radicals and their actions and hence they mitigate their harmful effects. The present study was designed to investigate pharmacological properties viz., antioxidant, antibacterial and antiproliferative activities of cinnamaldehyde and eugenol, the two naturally occurring phenylpropanoids present in Cinnamomum spp. and other plants.MethodsThe antioxidant potential of test compounds was evaluated by measuring DPPH free radical scavenging, reducing power and metal ion chelating activities. Protection against membrane damage was assayed by inhibition of lipid peroxidation in rat liver homogenate. Antibacterial activity was measured by Kirby-Bauer disc diffusion method while antiproliferative activity of test compounds was measured by sulforhodamine-B (SRB) assay.ResultsEugenol exhibited noticeable antioxidant potential in DPPH radical scavenging (81 %) and reducing power (1.12) assays at 1.0 μM/ml and 0.1 μM/ml concentrations, respectively. IC50 value of eugenol for radical scavenging activity was found to be 0.495 μM/ml. Cinnamaldehyde demonstrated considerable metal ion chelating ability (75 %) at 50 μM/ml and moderate lipo-protective activity in lipid peroxidation assay at 3 μM/ml. In addition cinnamaldehyde also showed appreciable antibacterial activity (zone of inhibition 32–42 mm) against Bacillus cereus (MTCC 6840), Streptococcus mutans (MTCC 497), Proteus vulgaris (MTCC 7299), Salmonella typhi (MTCC 3917) and Bordetella bronchiseptica (MTCC 6838) while eugenol produced moderate activity at 80 μM/disc. Cinnamaldehyde exhibited comparatively better antiproliferative potential against breast (T47D) and lung (NCI-H322) cancer cell lines than eugenol in SRB assay at 50 μM concentration.ConclusionCinnamaldehyde possessed metal ion chelating, lipo-protective, antibacterial and antiproliferative activities while eugenol showed potent H-atom donating potential indicating radical quenching and reducing power abilities. Medicinal attributes shown by both the compounds indicated their usefulness in food and pharmaceutical sector.

Measurement of Oxidation of Biomolecules and Inactivation of Bioparticles in Aqueous Media by Radicals Injected from Electrical Discharge Devices

ABSTRACTRecently, commercial home electric appliances with bactericidal/viral inactivation function of room air have been widely available. Most of these home appliances equip discharge devices which possibly generate reactive oxygen and nitrogen oxides. However, these effects are still controversial problem. To elucidate the inactivation effect of these devices at molecular level, we have been considering large DNA molecules as a biomarker to be used. By exposing DNA solution to the gas containing the chemical species, oxidative damage to DNA such as strand breaks can be induced. We have conducted the single-molecule-based evaluation method of DNA strand break using large linear DNA molecules. The obtained breakage rate could possibly be used for evaluation of reactivity of radicals generated by the discharge devices.

Biomolecules Oxidation and Antioxidant Enzymes Response as a Result of Injection of Oxidative Stressor into 5th Instar of Schistocerca Gregaria (Orthoptera, Acrididae)

Injection of heat killed Escherichia coli into the 5th instar of Schitocerca gregaria lead to deleterious effect on macromolecules such as proteins, lipids, and DNA, which expressed in the form of increased levels of carbonyls, peroxides, strand breaks, respectively. The results showed significant difference, in macromolecules damage, between control insect and injected one through time course and application of different concentration of E. coli. The present results were focusing on properties of lipopolysaccaride of bacterial cell wall which cause production of nitric oxide radical as an immune response of insect and also can react with superoxide anion radical that form peroxynitrite that considered as reactive nitrogen species which cause macromolecules damage Also, the results showed that the antioxidant enzymes activities, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were elevated over constitutive levels, in response to the injected oxidative stressor heat killed E. coli. The levels of both the constitutive and induced activities of GPx and are always higher in whole midgut homogenate than in thoracic muscles. The results may represent some sort of stressful conditions that may increase the vulnerability of insect pests to control measures.

Longevity of animals under reactive oxygen species stress and disease susceptibility due to global warming.

The world is projected to experience an approximate doubling of atmospheric CO2 concentration in the next decades. Rise in atmospheric CO2 level as one of the most important reasons is expected to contribute to raise the mean global temperature 1.4 °C-5.8 °C by that time. A survey from 128 countries speculates that global warming is primarily due to increase in atmospheric CO2 level that is produced mainly by anthropogenic activities. Exposure of animals to high environmental temperatures is mostly accompanied by unwanted acceleration of certain biochemical pathways in their cells. One of such examples is augmentation in generation of reactive oxygen species (ROS) and subsequent increase in oxidation of lipids, proteins and nucleic acids by ROS. Increase in oxidation of biomolecules leads to a state called as oxidative stress (OS). Finally, the increase in OS condition induces abnormality in physiology of animals under elevated temperature. Exposure of animals to rise in habitat temperature is found to boost the metabolism of animals and a very strong and positive correlation exists between metabolism and levels of ROS and OS. Continuous induction of OS is negatively correlated with survivability and longevity and positively correlated with ageing in animals. Thus, it can be predicted that continuous exposure of animals to acute or gradual rise in habitat temperature due to global warming may induce OS, reduced survivability and longevity in animals in general and poikilotherms in particular. A positive correlation between metabolism and temperature in general and altered O2 consumption at elevated temperature in particular could also increase the risk of experiencing OS in homeotherms. Effects of global warming on longevity of animals through increased risk of protein misfolding and disease susceptibility due to OS as the cause or effects or both also cannot be ignored. Therefore, understanding the physiological impacts of global warming in relation to longevity of animals will become very crucial challenge to biologists of the present millennium.

EVALUATION OF SERUM-FREE IRON AND GLYCATED HEMOGLOBIN IN UNCONTROLLED TYPE-II DIABETIC PATIENTS

ABSTRACTObjective: Diabetes mellitus (DM) is a common health problem in the world. Free iron may contribute to the pathogenesis and progress of thisdisease and its complication. Iron causes hyperinsulinemia by decreasing the insulin uptake and metabolism by hepatocytes. Elevated iron stores arecommonly found in insulin resistance. Iron in its free form is known to induce oxidation of biomolecules by producing harmful hydroxyl radicals. Inthis study, we aimed to estimate and compare the serum levels of free iron in diabetes and healthy individuals.Methods: This study included 244 subjects in two groups. Group-I comprised 204 subjects with DM and Group-II comprised 40 healthy subjects. Bloodsugar, free iron, and glycated hemoglobin were analyzed in blood samples using standard kits. The results of all the parameters were expressed asmean±standard deviation. Student t-test was done to assess the statistical significance between two groups. The association between the parameterswas studied by Pearson correlation.Results: In this study, we found a significant increase in serum free iron in Group-I (p&lt;0.01) when compared with Group-II. A significant correlationbetween the serum free iron and glycated hemoglobin (r=0.59; p&lt;0.001) and fasting blood sugar (r=0.43; p&lt;0.001).Conclusion: The elevated serum free iron in uncontrolled diabetes may contribute to oxidative stress which may be associated with complicationsof diabetes.Keywords: Free iron, Glycated hemoglobin, Hyperinsulinemia, Diabetes mellitus.

Redox proteomics: Methods for the identification and enrichment of redox-modified proteins and their applications.

PTMs are defined as covalent additions to functional groups of amino acid residues in proteins like phosphorylation, glycosylation, S-nitrosylation, acetylation, methylation, lipidation, SUMOylation as well as oxidation. Oxidation of proteins has been characterized as a double-edged sword. While oxidative modifications, in particular of cysteine residues, are widely involved in the regulation of cellular homeostasis, oxidative stress resulting in the oxidation of biomolecules along with the disruption of their biological functions can be associated with the development of diseases, such as cancer, diabetes, and neurodegenerative diseases, respectively. This is also the case for advanced glycation end products, which result from chemical reactions of keto compounds such as oxidized sugars with proteins. The role of oxidative modifications under physiological and pathophysiological conditions remains largely unknown. Recently, novel technologies have been established that allow the enrichment, identification, and characterization of specific oxidative PTMs (oxPTMs). This is essential to develop strategies to prevent and treat diseases that are associated with oxidative stress. Therefore this review will focus on (i) the methods and technologies, which are currently applied for the detection, identification, and quantification of oxPTMs including the design of high throughput approaches and (ii) the analyses of oxPTMs related to physiological and pathological conditions.

A Kinetic Platform to Determine the Fate of Hydrogen Peroxide in Escherichia coli.

Hydrogen peroxide (H2O2) is used by phagocytic cells of the innate immune response to kill engulfed bacteria. H2O2 diffuses freely into bacteria, where it can wreak havoc on sensitive biomolecules if it is not rapidly detoxified. Accordingly, bacteria have evolved numerous systems to defend themselves against H2O2, and the importance of these systems to pathogenesis has been substantiated by the many bacteria that require them to establish or sustain infections. The kinetic competition for H2O2 within bacteria is complex, which suggests that quantitative models will improve interpretation and prediction of network behavior. To date, such models have been of limited scope, and this inspired us to construct a quantitative, systems-level model of H2O2 detoxification in Escherichia coli that includes detoxification enzymes, H2O2-dependent transcriptional regulation, enzyme degradation, the Fenton reaction and damage caused by •OH, oxidation of biomolecules by H2O2, and repair processes. After using an iterative computational and experimental procedure to train the model, we leveraged it to predict how H2O2 detoxification would change in response to an environmental perturbation that pathogens encounter within host phagosomes, carbon source deprivation, which leads to translational inhibition and limited availability of NADH. We found that the model accurately predicted that NADH depletion would delay clearance at low H2O2 concentrations and that detoxification at higher concentrations would resemble that of carbon-replete conditions. These results suggest that protein synthesis during bolus H2O2 stress does not affect clearance dynamics and that access to catabolites only matters at low H2O2 concentrations. We anticipate that this model will serve as a computational tool for the quantitative exploration and dissection of oxidative stress in bacteria, and that the model and methods used to develop it will provide important templates for the generation of comparable models for other bacterial species.

A novel approach for oxidation analysis of therapeutic proteins

Measuring and monitoring of protein oxidation modifications is important for biopharmaceutical process development and stability assessment during long-term storage. Currently available methods for biomolecules oxidation analysis use time-consuming peptide mapping analysis. Therefore, it is desirable to develop high-throughput methods for advanced process control of protein oxidation. Here, we present a novel approach by which oxidative protein modifications are monitored by an indirect potentiometric method. The method is based on adding an electron mediator, which enhances electron transfer (ET) between all redox species and the electrode surface. Specifically, the procedure involves measuring the sharp change in the open circuit potential (OCP) for the mediator system (redox couple) as a result of its interaction with the oxidized protein species in the solution. Application of Pt and Ag/AgCl microelectrodes allowed for a high-sensitivity protein oxidation analysis. We found that the Ru(NH3)62+/3+ redox couple is suitable for measuring the total oxidation of a wide range of therapeutic proteins between 1.1 and 13.6%. Accuracy determined by comparing with the known percentage oxidation of the reference standard showed that percentage oxidation determined for each sample was within ±20% of the expected percentage oxidation determined by mass spectrometry.

Fungal weathering of asbestos in semi arid regions of India

The science of Geomicrobiology, which deals with mineral- microbe interaction in nature contributes effectively to three important processes namely- mineral and metal bioremediation, biomining and soil mineral formation by microbes. Bioremediation one of the important process of the above, degrades or transforms hazardous contaminants to less toxic compounds. Several groups of fungi have proved highly efficient in this aspect, with asbestos being one such toxic entity in the environment on which their activity was studied. The present investigation uses the same tool as a device for detoxifying asbestos, a potent carcinogenic entity; with fungal isolates native to the asbestos mines of Rajasthan, India, being investigated for the first time. The cellular mechanism of asbestos toxicity is mainly attributed to the presence of iron in its chemical composition which catalyzes generation of free radicals leading to oxidation of biomolecules. The two dominant novel species found therein, identified as Aspergillus tubingenesis and Coemansia reversa have proved capable of actively removing iron from asbestos fibers as studied by scanning electron microscopy- electron diffraction X-ray (SEM–EDX) analysis. This probably could lead to a reduction in toxicity of asbestos, due to reduced iron concentration as reported in related studies. Many fungi are known to release iron chelating compounds, siderophores, which could be instrumental in the study. The findings related to two new fungal species being added to the list of earlier identified fungal bioremediators of asbestos, widens the prospect of using bioremediation as an effective tool for asbestos detoxification

PP16 - Fluorescent microscopy study of intracellular distribution of carbonylated proteins and lipids

Carbonylation is an important oxidative modification of biomolecules and widely accepted as a biomarker of oxidative stress (OS). Carbonylated proteins, lipids, and nucleic acids have been intensively studied and were often linked to the onset or progression of OS related disorders. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization with specific chemical probes. Several analytical methods for identification and quantification of carbonylated biomolecules have been reported. However, in order to understand cellular carbonylation pathways and reveal their biological relevance it is crucial to study their intracellular formation and spatial distribution. Here, we used coumarin-hydrazide, a fluorescent chemical probe, for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The protocol was verified in a cellular model of paraquat induced OS and compared with a conventional DNPH-based immunocytochemistry. The specificity of coumarin-hydrazide towards carbonylated proteins and lipids was confirmed by a wide range of analytical techniques including gel electrophoresis, thin layer chromatography, and mass spectrometry. Additionally, co-distribution with oxidized lipids was evaluated by confocal microscopy using oxidized phosphatidylcholine specific natural antibodies. We have applied this method to detect carbonyls in several cellular models of oxidative stress including paracetamol induced hepatocyte toxicity and nitrosative stress in primary cardiomyocytes. A strong increase in biomolecule oxidation was observed in all studied OS models and most of the carbonylated species were accumulated in perinuclear space. Using confocal microscopy, co-localization of carbonylated biomolecules with endoplasmic reticulum was also demonstrated.

Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes.

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.

Electrochemical Sensitive Determination of Nanomolar Guanine from ZnO Nanorods Coated on Platinum Electrode

AbstractThe crystalline ZnO nanorods, grown on Si‐substrate (100) at a low temperature using a hydrothermal process were used for modification of platinum electrode using PVC and THF for in order to test their chemosensor activity. The modified electrode can be used for selective nanomolar determination of guanine in aqueous media without any interference from other biomolecules. The prepared chemosensor has a lower detection limit of 40 nM for guanine, and a modification of the electrodes with ZnO nanorods increases the reproducibility by reducing the fouling effect caused by the oxidation of biomolecules on the electrode surface as the prepared electrode can be used for more than 30 consecutive scans and can be used for 5 days without any change in current intensity or sensitivity.

Photoinactivation of tyrosinase sensitized by folic acid photoproducts

Tyrosinase catalyzes in mammals the first and rate-limiting step in the biosynthesis of the melanin, the main pigment of the skin. Pterins, heterocyclic compounds able to photoinduce oxidation of biomolecules, accumulate in the skin of patients suffering from vitiligo, where there is a lack of melanin. Folic acid (PteGlu) is a conjugated pterin widespread in biological systems. Aqueous solutions of tyrosinase were exposed to UV-A irradiation (350nm) in the presence of PteGlu and its photoproducts (6-formylpterin and 6-carboxypterin). The reactions were followed by UV–Vis spectrophotometry, enzyme activity measurement, fluorescence spectroscopy and HPLC. In this work, we present data that demonstrate unequivocally that solutions of tyrosinase exposed to UV-A irradiation in the presence of PteGlu, undergo enzyme inactivation. However, PteGlu itself causes a negligible effect on the activity of the enzyme. In contrast, PteGlu photoproducts are efficient photosensitizers. The tyrosinase inactivation involves two different pathways: (i) a photosensitization process and (ii) the oxidation of the enzyme by the hydrogen peroxide produced during the photooxidation of PteGlu and its photoproduct. The former pathway affects both the active site and the tryptophan residues, whereas the latter affects only the active site. The biological implications of the results are discussed.