Investigation of the influence of pH and temperature on melanization and survival under oxidative stress in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.

Effects of radical scavengers for reactive oxygen species on vitamin K-induced phototoxicity under UVA irradiation.

Vitamin K possesses efficacy as a topical dermatological agent. However, vitamin K is phototoxic and susceptible to photodegradation. Herein, we investigated the mechanisms underlying the phototoxicity of phylloquinone (PK, vitamin K1) and menaquinone-4 (MK-4, vitamin K2) under ultraviolet A (UVA) irradiation using various reactive oxygen species (ROS) scavengers. This resulted in the production of superoxide anion radicals via type I and singlet oxygen via type II photodynamic reactions, which were quenched by the ROS scavengers: superoxide dismutase and sodium azide (NaN3). In HaCaT cells, MK-4 and PK induced the production of intracellular ROS, particularly hydrogen peroxide, in response to UVA irradiation. Furthermore, the addition of catalase successfully decreased maximum ROS levels by approximately 30%. NaN3 and catalase decreased the maximum reduction in cell viability induced by UVA-irradiated PK and MK-4 in cell viability by approximately 2-7-fold. Additionally, ROS scavengers had no effect on the photodegradation of PK or MK-4 at 373 nm. Therefore, the phototoxicities of PK and MK-4 were attributed to the generation of singlet oxygen and hydrogen peroxide, underscoring the importance of photoshielding in circumventing phototoxicity.

Secretory Production of the Hericium erinaceus Laccase from Saccharomyces cerevisiae.

Mushroom laccases play a crucial role in lignin depolymerization, one of the most critical challenges in lignin utilization. Importantly, laccases can utilize a wide range of substrates, such as toxicants and antibiotics. This study isolated a novel laccase, named HeLac4c, from endophytic white-rot fungi Hericium erinaceus mushrooms. The cDNAs for this enzyme were 1569 bp in length and encoded a protein of 523 amino acids, including a 20 amino-acid signal peptide. Active extracellular production of glycosylated laccases from Saccharomyces cerevisiae was successfully achieved by selecting an optimal translational fusion partner. We observed that 5 and 10 mM Ca2+, Zn2+, and K+ increased laccase activity, whereas 5 mM Fe2+ and Al3+ inhibited laccase activity. The laccase activity was inhibited by the addition of low concentrations of sodium azide and L-cysteine. The optimal pH for the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was 4.4. Guaiacylglycerol-ß-guaiacyl ether, a lignin model compound, was polymerized by the HeLac4c enzyme. These results indicated that HeLac4c is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products for environmental biotechnological applications.

Evaluating the Stability of Lytic and Lysogenic Bacteriophages in Various Protectants.

Phage therapy has regained value as a potential alternative and a complementary anti-infective approach to antibiotics in the fight against bacterial pathogens. Due to their host specificity, non-pathogenic nature for humans, and low production cost, phages offer an effective opportunity for utilization in healthcare, agriculture, and food preservation. Well-defined storage conditions are essential for commercialization and dissemination of phage usage. For this purpose, in our study, after the isolation and characterization of two different phages, one lytic and the other lysogenic; storage and shelf-life studies of phages were evaluated in a presence of various protectants (glycerol, sodium azide, DMSO with chloroform) and without any protectant during 8-month period at four different temperatures. The short-time stability of the lytic P. syringae phage and lysogenic MRSA phage, which were determined by STEM analysis to belong to the Straboviridae and Siphoviridae families, respectively were also examined for the different temperatures and the pH levels ranging from 1.0 to 14.0. This study revealed the storage-model of phages that exhibit distinct lifecycles, for the first time and provided a theoretical basis for development and application of phages, has yielded valuable findings contributing to understanding of phage biology.

A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties.

The gene for a previously unexplored two-domain laccase was identified in the genome of actinobacterium Streptomyces carpinensis VKM Ac-1300. The two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike the most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. Half-inactivation time of ScaSL at 80°C was 40 min. The laccase was able to oxidize a non-phenolic organic compound ABTS at a maximum rate at pH 4.7, and to oxidized a phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. The laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was slightly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, the laccase was completely inhibited by 100 mM sodium azide. The determined Km and kcat of the enzyme for ABTS were 0.1 mM and 20 s-1, respectively. The Km and kcat for 2,6-dimethoxyphenol were 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed polymerization of humic acids and lignin. Redox potential of the laccase was 0.472 ± 0.007 V. Thus, the ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. Crystal structure of ScaSL was determined with 2.35 Å resolution. Comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.

Rapid and simple identification of trace amounts of sodium azide in beverages and bodily fluids followed by derivatization and liquid chromatography-electrospray ionization tandem mass spectrometry.

RATIONALE: Sodium azide (NaN3 ) is a toxic chemical agent to humans by ingestion and inhalation with a growing number of intentional exposures and accidental cases over the last few decades. Due to its low molecular weight and lack of any chromophore, its retention and detection by reverse-phase liquid chromatography-ultraviolet-mass spectrometry methods are a challenging task. METHODS: To be able to confirm azide exposure, we have developed a method to identify azide in both beverages and bodily fluids. The identification of azide (N3 - ) is based on derivatization with N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) at 25°C for 15 min followed by LC/ESI-MS/MS analysis, with no other sample preparation. RESULTS: The azide after derivatization (CAX-N3 ) was stable, retainable by LC and sensitively detected by selected reaction monitoring. The ESI-MS/MS fragmentation of the M+ precursor ion produced characteristic product ions at m/z 118, 100, 91 and 86. The calibration curves for CAX-N3 showed linearity over two orders of magnitude with R2 value of 0.99. Low limits of identification of 0.1-0.5 ng/mL were obtained in all investigated matrices (drinking water, tea, orange juice, plasma and urine). CONCLUSIONS: Compared with previously reported chromatography-based methods, this method that was based on derivatization and LC/ESI-MS/MS analysis was substantially more sensitive, simpler and faster. The method can be used for forensic investigation to confirm azide exposure from fatal use to much smaller intoxication dose.

Effectiveness of curcumin-based antimicrobial photodynamic therapy against Staphylococcus aureus.

PURPOSE: This study investigated the effectiveness of curcumin-based antimicrobial photodynamic therapy (aPDT) against Staphylococcus aureus (S. aureus), the causative agent of ventilator-associated pneumonia. METHODS: Curcumin was added to S. aureus culture medium at concentrations of 25, 2.5, and 0.25 µM. After 60 min (20-25°C), each culture was irradiated for 1 and 3 min, and viable bacteria were counted. Curcumin (25 µM) was also added to a bacterial suspension with D-mannitol and sodium azide; microbial counts were determined after irradiation for 3 min. RESULTS: S. aureus was significantly reduced in the 1-min (P = 0.043) and 3-min (P = 0.011) irradiation groups in comparison to the 0-min irradiation group with 25 µM curcumin. No significant differences were observed between the curcumin alone group and the curcumin plus D-mannitol or sodium azide group. CONCLUSION: The findings of this study indicate that prolonged exposure (≥1 min) of S. aureus to LED in 25 µM curcumin solution induces cell wall injury. Curcumin-based aPDT as an adjunct to conventional oral care, employing existing dentistry equipment, offers a promising approach that does not rely on antimicrobial drugs or allows the emergence of resistant bacterial strains. This suggests its potential role in future strategies aimed at preventing ventilator-associated pneumonia.

Hypoxia-induced mitochondrial stress granules.

Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining hypoxia-induced mitochondrial protein aggregation in C. elegans, we found that sublethal hypoxia, sodium azide, or heat shock-induced abundant ethidium bromide staining mitochondrial granules that preceded evidence of protein aggregation. Genetic manipulations that reduce cellular and organismal hypoxic death block the formation of these mitochondrial stress granules (mitoSG). Knockdown of mitochondrial nucleoid proteins also blocked the formation of mitoSG by a mechanism distinct from the mitochondrial unfolded protein response. Lack of the major mitochondrial matrix protease LONP-1 resulted in the constitutive formation of mitoSG without external stress. Ethidium bromide-staining RNA-containing mitochondrial granules were also observed in rat cardiomyocytes treated with sodium azide, a hypoxia mimetic. Mitochondrial stress granules are an early mitochondrial pathology controlled by LONP and the nucleoid, preceding hypoxia-induced protein aggregation.

Assessment of Bio-physiological damages and cytological aberrations in cowpea varieties treated with gamma rays and sodium azide.

The assessment of mutagen induced biological damage forms an important study in determining the mutagenic potency and genotypic sensitivity, a vital aspect in mutation breeding programs. A prior assessment of lethal dose (LD50), mutagen induced biological damage (alterations in bio-physiological traits and frequency of cytological aberrations) is a prerequisite for determining an optimum mutagen dose in a successful mutation breeding experiment. Therefore, in a multi-year project of mutation breeding, two widely cultivated varieties of cowpea viz., Gomati VU-89 and Pusa-578, were treated with gamma (γ) rays and sodium azide (SA) doses. The results reflected a proportionate increase in bio-physiological damages with the increase in mutagenic doses and caused a substantial reduction in mean seed germination and seedling height. Different cytological aberrations such as cytomixis, univalents, chromosome stickiness, precocious separation, unequal separation, bridges, laggards, disturbed polarity, dyads, triads, and polyads were observed in both varieties. All the mutagen doses induced a broader spectrum of cytological aberrations with varying frequencies.

Clinical features of COVID-19 rapid antigen test exposures reported by an Australian poisons information centre: a prospective study.

INTRODUCTION: Coronavirus disease COVID-19 rapid antigen tests are a useful tool in detecting infection, and their use has increased in many countries since they became commercially available in late 2021. Some rapid antigen tests contain sodium azide, which can be toxic in small doses. This study aimed to describe the clinical characteristics of exposures to COVID-19 rapid antigen tests. METHODS: This is a prospective study conducted by the New South Wales Poisons Information Centre. From 22 January 2022 to 31 August 2022, rapid antigen test exposures were followed up to obtain outcome information. Data collected included: brand/ingredients, exposure route, demographics, symptoms, and disposition. RESULTS: We recorded 218 exposures in the seven-month study period. Complete follow-up information was available in 75% (n = 164). There were 53 exposures to sodium azide-containing products (35 with follow-up data) and 165 to non-sodium azide-containing products and unknown ingredient exposures (129 with follow-up data). Overall, unintentional exposures predominated (n = 182), and 151 were ingestions. The vast majority (>90%) did not develop symptoms, and all symptoms that developed were mild. Most cases (95%, n = 208) did not require referral to a healthcare facility. CONCLUSIONS: In this prospective series, few patients developed symptoms, regardless of the sodium azide content, likely due to low concentration and low volume within the test kits. However, ongoing toxicovigilance is warranted.

Selective photodynamic effects on cervical adenocarcinoma cells provided by F127 Pluronic®-based micelles modulating hypericin delivery

Abstract Cervical cancer is a leading cause of death among women. The endocervical adenocarcinoma (ECA) represents an aggressive and metastatic type of cancer with no effective treatment options currently available. We evaluated the antitumoral and anti-migratory effects of hypericin (HYP) encapsulated on Pluronic F127 (F127/HYP) photodynamic therapy (PDT) against a human cell line derived from invasive cervical adenocarcinoma (HeLa) compared to a human epithelial cell line (HaCaT). The phototoxicity and cytotoxicity of F127/HYP were evaluated by the following assays: colorimetric assay, MTT, cellular morphological changes by microscopy and long-term cytotoxicity by clonogenic assay. In addition, we performed fluorescence microscopy to analyze cell uptake and subcellular distribution of F127/HYP, cell death pathway and reactive oxygen species (ROS) production. The PDT mechanism was determined with sodium azide and D-mannitol and cell migration by wound-healing assay. The treatment with F127/HYP promoted a phototoxic result in the HeLa cells in a dose-dependent and selective form. Internalization of F127/HYP was observed mainly in the mitochondria, causing cell death by necrosis and ROS production especially by the type II PDT mechanism. Furthermore, F127/HYP reduced the long-term proliferation and migration capacity of HeLa cells. Overall, our results indicate a potentially application of F127/HYP micelles as a novel approach for PDT with HYP delivery to more specifically treat ECA.

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

N-myc downstream regulated gene 1 (ndrg1) functions as a molecular switch for cellular adaptation to hypoxia.

Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation, which may preserve ATP in the kidney and ionocytes and contribute to energy homeostasis. Lastly, we show that sodium azide treatment, which increases lactate levels under normoxia, is sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a is essential for hypoxia adaptation and functions downstream of lactate signaling to induce NKA degradation, a process known to conserve cellular energy.

Is In-Service Granular Activated Carbon Biologically Active? An Evaluation of Alternative Experimental Methods to Distinguish Adsorption and Biodegradation in GAC.

In-service granular activated carbon (GAC) may transform into biological activated carbon (BAC) and remove contaminants through both adsorption and biodegradation, but it is difficult to determine its biodegradative capacity. One approach to understand the GAC biodegradative capacity is to compare the performance between unsterilized and sterilized GAC, but the sterilization methods may not ensure effective microbial inhibition and may affect adsorption. This study identified the 14C-glucose respiration rate as the best metric to evaluate the effectiveness of three sterilization methods: sodium azide addition, autoclaving, and γ irradiation. The sterilization protocols were refined, including continuously feeding 300 mg/L of sodium azide, three cycles of autoclaving, and 10-12 kGy of γ irradiation. Parallel minicolumn tests were conducted to identify sodium azide addition as the most broadly effective sterilization method with an insignificant effect on adsorption in most cases, except for the adsorption of anionic compounds under certain conditions. Nevertheless, this problem was solved by decreasing the azide dosage as long as it is still sufficient to provide effective microbial inhibition. This study helps to develop an approach that differentiates adsorption and biodegradation in GAC, which could be used by future studies to advance our understanding of BAC filtration.

Bis(oxiranes) Containing Cyclooctane Core: Synthesis and Reactivity towards NaN3.

Reactions of oxirane ring opening provide a powerful tool for regio- and stereoselective synthesis of polyfunctional and heterocyclic compounds, widely used in organic chemistry and drug design. Cyclooctane, alongside other medium-sized rings, is of interest as a novel molecular platform for the construction of target-oriented leads. Additionally, cyclooctane derivatives are well known to be prone to transannular reactions, which makes them a promising object in the search for novel approaches to polycyclic structures. In the present work, a series of cyclooctanediones was studied in Corey-Chaykovsky reactions, and novel spirocyclic bis(oxiranes) containing cyclooctane core, namely, 1,5-dioxadispiro[2.0.2.6]dodecane and 1,8-dioxadispiro[2.3.2.3]dodecane, were synthesized. Ring opening of the obtained bis(oxiranes) upon treatment with sodium azide was investigated, and it was found that the reaction path is determined by the reciprocal orientation of oxygen atoms in the oxirane moieties. Diastereomers of the bis(oxiranes) with cis-orientation underwent independent ring opening, supplying corresponding diazidodiols, while in the case of stereoisomers with trans-orientation, domino-like reactions occurred, including intramolecular nucleophilic attack and the formation of a novel three- or six-membered O-containing ring. Summarily, a straightforward approach to polyfunctional compounds containing cyclooctane or oxabicyclo[3.3.1]nonane cores, employing bis(oxiranes), was elaborated.

New ecofriendly heterogeneous nano-catalyst for the synthesis of 1-substituted and 5-substituted 1H-tetrazole derivatives.

A novel ecofriendly heterogeneous catalyst containing Schiff base coordinated Cu(II) covalently attached to Fe3O4@SiO2 nanoparticles through imidazolium linker [Fe3O4@SiO2-Im(Br)-SB-Cu (II)] was synthesized and characterized by using various techniques. The catalytic efficiency of this nano-catalyst was tested in water in the synthesis of tetrazole derivatives using two one-pot multicomponent reaction (MCR) models: The synthesis of 1-aryl 1H-tetrazole derivatives from the reaction of aniline, triethyl orthoformate, and sodium azide and the synthesis of 5-aryl 1H-tetrazole derivatives from the reaction of benzaldehyde, hydroxy amine hydrochloride, and sodium azide. The investigation showed that (i) The catalyst is highly efficient in the synthesis of tetrazole derivatives with high yield (97%) in aqueous medium and mild temperatures; (ii) The catalytic effectiveness is due to the synergy between the metallic center and the imidazolium ion and (iii) The reuse advantage of the catalyst without contamination or significant loss (12% of loss range) in the catalytic activity.

Effect of Sodium Azide on Yield and its Components in Bread Wheat (Triticum aestivum L.).

<b>Background and Objective:</b> The wheat crop is considered one of the most important crops globally, especially in Egypt. It has great nutritional importance, so it was necessary to increase productivity and any genetic improvement depends on the presence of many genetic differences so that breeders can achieve this. This study aimed to use chemical mutagenic (sodium azide) to obtain the desired genetic differences in two wheat cultivars. <b>Materials and Methods:</b> Two types of bread Sids 12 and Giza 164 were treated with different concentrations of sodium azide (NaN₃) (1000, 2000, 3000, 4000, 5000 and 6000 ppm). <b>Results:</b> The highest grain/plant 78.91 g was obtained from Sis12 and 62.96 g from Giza 164 compared to the control 42.57 and 40.24 g for Sids 12 and Giza 164, respectively. Also from the results obtained, the relationship of yield was positive and significant with both grain/spike, spikelet's no./spike spikes no./plant and height/plant. On the contrary, it was negative and significant with a 1000-grain weight (-0.433). <b>Conclusion:</b> The two treatments (1000 and 2000 ppm) were the best in the Sids 12, while (1000 and 5000 ppm) were the best treatments in the Giza 164.

Endocytosis-like DNA uptake by cell wall-deficient bacteria.

Horizontal gene transfer in bacteria is widely believed to occur via conjugation, transduction and transformation. These mechanisms facilitate the passage of DNA across the protective cell wall using sophisticated machinery. Here, we report that cell wall-deficient bacteria can engulf DNA and other extracellular material via an endocytosis-like process. Specifically, we show that L-forms of the filamentous actinomycete Kitasatospora viridifaciens can take up plasmid DNA, polysaccharides (dextran) and 150-nm lipid nanoparticles. The process involves invagination of the cytoplasmic membrane, leading to formation of intracellular vesicles that encapsulate extracellular material. DNA uptake is not affected by deletion of genes homologous to comEC and comEA, which are required for natural transformation in other species. However, uptake is inhibited by sodium azide or incubation at 4 °C, suggesting the process is energy-dependent. The encapsulated materials are released into the cytoplasm upon degradation of the vesicle membrane. Given that cell wall-deficient bacteria are considered a model for early life forms, our work reveals a possible mechanism for primordial cells to acquire food or genetic material before invention of the bacterial cell wall.

Mutagenic effects of sodium azide on in vitro mutagenesis, polymorphism and genomic instability in wheat (Triticum aestivum L.).

INTRODUCTION: Breeding studies are commonly conducted to develop new cultivars with high yield levels and improved quality traits. Chemically-induced mutations are used to create genetic variations in wheat genomes. Various physical and chemical mutagens are used to increase frequency of mutations and facilitate the selection processes. Sodium azide (SA) is largely employed to induce mutations of the genes regulating essential traits. Such mutations may also elucidate gene functions of the mutant phenotypes. Present experiments were conducted to investigate potential use of conventional chemical mutagenesis technique through SA for mature embryo culture in wheat. METHODS AND RESULTS: Sodium azide mutagenesis was experimented with 4 treatment durations (1, 2, 3 and 4 h) and 5 treatment concentrations (0, 1, 2, 3 and 4 mM). Mature embryos were subjected to experimental treatments to detect optimum doses of mutagenesis and to estimate polymorphism and genomic instability. Primarily, 50% reduction in number of regenerated plants as compared to the control (LD50) was adopted as the optimum dose. Based on LD50 criterion, the optimum value was achieved at 1 h duration of 4 mM SA concentration. Afterwards, inter-primer binding site markers were applied to investigate polymorphism and genomic instability in the regenerated plants. CONCLUSIONS: Present findings revealed that efficiency of chemical mutagenesis could be improved through the use of molecular technology and such mutations may assist plant breeders in developing high-yield cultivars.

Photo-oxidation and Photoreduction of Catechols by Chlorophyll Metabolites and Methylene Blue.

While plant-derived oxidants can protect cells from oxidative damage, limited research has examined the role of dietary chlorophyll. Photoreduction of ubiquinone by chlorophyll metabolites and red light has been reported in vitro and in animal models. Herein we examined photo-oxidation and photoreduction reactions of catechols, dopamine and hydrocaffeic acid. Photo-oxidation of dopamine by methylene blue and the chlorophyll metabolites pheophorbide A, chlorin e6 and sodium copper chlorophyllin was studied by monitoring aminochrome, the cyclized product of the dopamine o-quinone with its amine. Singlet oxygen scavengers including sodium azide, ascorbate and glutathione decreased aminochrome formation by methylene blue and pheophorbide A. Addition of EDTA, a tertiary amine electron donor, to the reaction of dopamine, photosensitizer and red light decreased aminochrome formation. Photoreduction of the dopamine o-quinone produced by mushroom tyrosinase was achieved by both methylene blue and pheophorbide A only when an electron donor was included. Due to limited solubility, photo-oxidation and photoreduction reactions by pheophorbide A required 5-7.5% dimethylformamide for optimal reactivity. Catalytic photoreduction of 2,3-dimethoxy-5-methyl-p-benzoquinone by methylene blue or pheophorbide A and tertiary amine electron donors was observed. Among the chlorophyll metabolites, pheophorbide A was more effective than chlorin e6 or sodium copper chlorophyllin in photo-oxidation of dopamine and photoreduction reactions. Singlet oxygen inhibited lactate dehydrogenase A activity, and higher molecular weight protein cross-links were observed on SDS-PAGE. Hydrocaffeic acid competed with lactate dehydrogenase A for reaction with singlet oxygen produced by methylene blue; however, no protection by hydrocaffeic acid (HCA) was observed when pheophorbide A was used. Cysteine modification of lactate dehydrogenase A by the o-quinone of hydrocaffeic acid was detected using a redox cycling stain. Inclusion of an electron donor decreased protein labeling.