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.

Biochemical and molecular profiling of induced high yielding M3 mutant lines of two Trigonella species: Insights into improved yield potential.

Trigonella, commonly known as Fenugreek, is among the most promising medicinal herbs consumed worldwide due its protein rich dietary contributions. This study involved induced mutagenesis on two Trigonella species (Trigonella foenum-graecum var. PEB and Trigonella corniculata var. Pusa kasuri) using caffeine and sodium azide as mutagens, resulting in the identification of nine high-yielding mutant lines in the M3 generation. Molecular characterization using SCoT markers revealed a high polymorphism of 28.3% and 46.7% in PEB and Pusa kasuri, respectively, facilitating the investigation of genetic divergence among the control and mutant lines. Similarity correlation analysis indicated a high similarity between mutant A and mutant C (0.97) and between mutant J and mutant O (0.88), while the lowest similarity was observed between mutant B and mutant F (0.74) and between control and mutant L (0.58). Mutant F and Mutant J displayed the highest seed yield and its attributing traits, and seed protein content in PEB and Pusa kasuri, respectively. Physiological parameters, including chlorophyll content (Mutants A and N) and carotenoids (mutant A and J), exhibited improvements. Assessment of stomatal and seed characteristics using scanning electron microscopy may lead to improved physiological processes and distinction at the interspecific level, respectively. Methanolic extracts of the control and the mutant lines of both species were subjected to GC-MS analysis, revealing 24 major phytocompounds known for their pharmacological activities (antioxidant, anti-inflammatory, anticancer, etc.). Statistical methods such as Pearson correlation heatmap and pairwise scatter plot matrix provided insights into the correlations and linear associations among parameters for both PEB and Pusa kasuri. The strong correlation between iron content and seeds per pod in the mutant lines suggests a promising avenue for further research. Continued research and breeding efforts using these mutants can lead to significant advancements in agriculture and medicine, benefiting farmers, consumers, and industries alike.

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.

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.

Discrimination of Receptor-Mediated Endocytosis by Surface-Enhanced Raman Scattering.

Cellular energy required for the maintenance of cellular life is stored in the form of adenosine triphosphate (ATP). Understanding cellular mechanisms, including ATP-dependent metabolisms, is crucial for disease diagnosis and treatment, including drug development and investigation of new therapeutic systems. As an ATP-dependent metabolism, endocytosis plays a key role not only in the internalization of molecules but also in processes including cell growth, differentiation, and signaling. To understand cellular mechanisms including endocytosis, many techniques ranging from molecular approaches to spectroscopy are used. Surface-enhanced Raman scattering (SERS) is shown to provide valuable label-free molecular information from living cells. In this study, receptor-mediated endocytosis was investigated with SERS by inhibiting endocytosis with ATP depletion agents: sodium azide (NaN3) and 2-deoxy-d-glucose (dG). Human lung bronchial epithelium (Beas-2b) cells, normal prostate epithelium (PNT1A) cells, and cervical cancer epithelium (HeLa) cells were used as models. First, the effect of NaN3 and dG on the cells were examined through cytotoxicity, apoptosis-necrosis, ATP assay, and uptake inhibition analysis. An attempt to relate the spectral changes in the cellular spectra to the studied cellular events, receptor-mediated endocytosis inhibition, was made. It was found that the effect of two different ATP depletion agents can be discriminated by SERS, and hence receptor-mediated endocytosis can be tracked from single living cells with the technique without using a label and with limited sample preparation.

Mitochondrial Inhibition by Sodium Azide Induces Assembly of eIF2α Phosphorylation-Independent Stress Granules in Mammalian Cells.

Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN3) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN3-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN3-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN3-induced SGs.

Nigella sativa callus treated with sodium azide exhibit augmented antioxidant activity and DNA damage inhibition.

Nigella sativa L. (NS) is an herbaceous plant, possessing phytochemicals of therapeutic importance. Thymoquinone is one of the active phytochemicals of NS that confers noteworthy antioxidant properties. Sodium azide, an agent of abiotic stress, can modulates antioxidant system in plants. In the present investigation, sodium azide (0, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM and 200 µM) doses administered to the in vitro NS callus cultures for production/modification of secondary metabolites with augmented activity. 200 µM sodium azide treated NS callus exhibited maximum peroxidase activity (1.286 ± 0.101 nanokatal mg-1 protein) and polyphenol oxidase activity (1.590 ± 0.110 nanokatal mg-1 protein), while 100 µM sodium azide treated NS callus for optimum catalase activity (1.250 ± 0.105 nanokatal mg-1 protein). Further, 200 µM sodium azide treated NS callus obtained significantly the highest phenolics (3.666 ± 0.475 mg g-1 callus fresh weight), 20 µM sodium azide treated NS callus, the highest flavonoids (1.308 ± 0.082 mg g-1 callus fresh weight) and 100 µM sodium azide treated NS callus, the highest carotenes (1.273 ± 0.066 mg g-1 callus fresh weight). However, NS callus exhibited a decrease in thymoquinone yield/content vis-à-vis possible emergence of its analog with 5.3 min retention time and an increase in antioxidant property. Treatment with 200 µM sodium azide registered significantly the lowest percent yield of callus extract (4.6 ± 0.36 mg g-1 callus fresh weight) and thymoquinone yield (16.65 ± 2.52 µg g-1 callus fresh weight) and content (0.36 ± 0.07 mg g-1 callus dry weight) and the highest antioxidant activity (3.873 ± 0.402%), signifying a negative correlation of the former with the latter. DNA damage inhibition (24.3 ± 1.7%) was recorded significantly maximum at 200 µM sodium azide treatment. Sodium azide treated callus also recorded emergence of a new peak at 5.3 min retention time (possibly an analog of thymoquinone with augmented antioxidant activity) whose area exhibits significantly negative correlation with callus extract yield and thymoquinone yield/content and positive correlation with antioxidant activity and in vitro DNA damage inhibition. Thus, sodium azide treatment to NS callus confers possible production of secondary metabolites or thymoquinone analog (s) responsible for elevated antioxidant property and inhibition to DNA damage. The formation of potent antioxidants through sodium azide treatment to NS could be worthy for nutraceutical and pharmaceutical industries.

Effect of Sodium Azide on Quantitative and Qualitative Stem Traits in the M2 Generation of Ethiopian Sesame (Sesamum indicum L.) Genotypes.

The emerging oilseed crop Sesamum indicum, also known as the queen of oilseeds, is being grown globally for its oil content for medicinal and nutritional values. One of the key challenges of sesame cultivation is its low productivity. In the present study, sodium azide (NaN3) was used as a chemical mutagen. The aim of this study was to examine the effect of NaN3 on quantitative and qualitative stem traits in the M2 generation of Ethiopian sesame (Sesamum indicum L.) genotypes. Seeds of fourteen sesame genotypes were used in this study and germinated and grown under greenhouse conditions. Different qualitative and quantitative data were collected and analyzed. Traits such as plant height, ground distance to first distance, and internode length were significantly affected by NaN3 treatment. The highest plant height was recorded in the control on Humera 1 and Baha Necho genotypes, while the lowest was observed on Setit 2 and Hirhir treated with the chemical. The highest ground distance to the first branch was observed in Gumero, while the least ground distance was recorded in Setit 1 in the treated and control genotypes, respectively. The best internode length was recorded on Setit 2 and ADI in the control, while the lowest internode length was observed in Setit 1 genotype treated with sodium azide. Genotypes such as ACC44, ADI, Baha Necho, Borkena, Gonder 1, and Setit 1 treated with NaN3 have showed glabrous type of stem hairiness. All the fourteen genotypes (both treated and control) were clustered into four groups. In conclusion, we observed a highly significant variation among the genotypes due the effect of the chemical and genotypes themselves. Hence, this report would create more genetic diversity for further sesame genetic research improvements.

Cisplatin-Induced Giant Cells Formation Is Involved in Chemoresistance of Melanoma Cells.

Melanoma is notoriously resistant to current cancer therapy. However, the chemoresistance mechanism of melanoma remains unclear. The present study unveiled that chemotherapy drug cisplatin induced the formation of giant cells, which exhibited enlargement in cell diameter and nucleus in mice and human melanoma cells. Giant cells were positive with melanoma maker S100 and cancer stem cell markers including ABCB5 and CD133 in vitro and in vivo. Moreover, giant cells retained the mitotic ability with expression of proliferation marker Ki-67 and exhibited multiple drug resistance to doxorubicin and actinomycin D. The mitochondria genesis/activities and cellular ATP level were significantly elevated in giant cells, implicating the demand for energy supply. Application of metabolic blockers such as sodium azide or 2-deoxy glucose abolished the cisplatin-induced giant cells formation and expression of cancer stemness markers. The present study unveils a novel chemoresistance mechanism of melanoma cells via size alteration and the anti-neoplastic strategy by targeting giant cells.

Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine.

Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.

Aqueous Humor Outflow Requires Active Cellular Metabolism in Mice.

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<<0.001), but reducing temperature did not affect ATP. Conclusions: Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac.

An extracellular laccase enzyme secreted from Sphingobacterium ksn-11 was purified to electrophoretic homogeneity, showing a molecular weight of 90 kDa. The purified enzyme was monomeric in nature confirmed by sodium dodecyl gel electrophoresis. The optimum temperature and pH were found to be 40 °C and 4.5 respectively. The enzyme showed highest substrate specificity for 2,2 azino-bis (ethylthiozoline-6-sulfonate) (ABTS), followed by syringaldazine. The Km value for ABTS was 2.12 mM with a Vmax value of 33.33 U/mg which was higher when compared with syringaldazine and guaiacol substrates. Sodium azide and EDTA inhibited the activity by 30%, whereas presence of Ca2+ and iron increased activity by 50%. The purified enzyme was immobilized in sodium alginate-silicon dioxide-polyvinyl alcohol beads and evaluated for diclofenac transformation studies. LC-MS analysis confirmed that immobilized laccase transformed diclofenac to 4-OH diclofenac after 4 h of incubation. 45 % of diclofenac was able to transform even at 3rd cycle of immobilized laccase use. Therefore, immobilized laccase can be used to transform or degrade several recalcitrant compounds from industrial effluents.

Iron is a ligand of SecA-like metal-binding domains in vivo.

The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

Composition-based prediction and rational manipulation of prion-like domain recruitment to stress granules.

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence.

Novel polymeric microspheres: Synthesis, enzyme immobilization, antimutagenic activity, and antimicrobial evaluation against pathogenic microorganisms.

New polymeric microspheres containing azomethine (1a-1c and 2a-2c) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT-IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis-Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (Vmax ) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well-diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.

NLRX1 Regulation Following Acute Mitochondrial Injury.

Several metabolic, cardiovascular, and neurological disorders are characterized by mitochondrial dysfunction followed by dysregulation of cellular energetics. Mitochondria play an important role in ATP production and cell death regulation. NLRX1, a mitochondria-targeted protein, is known to negatively regulate innate immunity, and cell death responses. However, the role of this protein in cellular homeostasis following mitochondrial injury is not well-understood. To understand the mechanisms underlying the effect of acute injury in regulating NLRX1 signaling pathways, we used an in vitro model of mitochondrial injury wherein, rat pulmonary microvascular endothelial cells were subjected to sodium azide treatment or glucose starvation. Both sodium azide and glucose starvation activated NF-κB and TBK1 associated innate immune response. Moreover, increased TBK1, IKK, IκB, and TRAF6 were recruited to mitochondria and interacted with NLRX1. Depletion of endogenous NLRX1 resulted in exacerbated NF-κB and TBK1 associated innate immune response and apoptosis. Our results suggest that NLRX1 participates in the regulation of innate immune response in mitochondria, and plays an important role in the maintenance of cellular homeostasis following acute mitochondrial injury. We propose that the mitochondrial recruitment of inflammatory mediators and their interaction with NLRX1 are protective responses to maintain cellular homeostasis following injury.

Creatine is Neuroprotective to Retinal Neurons In Vitro But Not In Vivo.

Purpose: To investigate the neuroprotective properties of creatine in the retina using in vitro and in vivo models of injury. Methods: Two different rat retinal culture systems (one containing retinal ganglion cells [RGC] and one not) were subjected to either metabolic stress, via treatments with the mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N-methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 nmol) or high IOP-induced ischemia reperfusion. Results: Creatine significantly prevented neuronal death induced by sodium azide and NMDA in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. Conclusions: Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation.

Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia.

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.