Formaldehyde Treatment Using Overexpressed Aldehyde Dehydrogenase 6 from Recombinant Saccharomyces cerevisiae.

Formaldehyde is a toxic compound due to its ability to react with proteins, nucleic acids and lipids and is the primary cause of nasopharyngeal cancer and sick building syndrome (SBS). Aldehyde dehydrogenases (ALDHs) are able to oxidize aldehyde substrates and maintain cellular homeostasis by metabolic reactions in prokaryotic and eukaryotic cells. ALDHs catalyze the conversions of various aldehydes to carboxylic acids using NAD or NADP as a cofactor. In this study, we designed a method for using aldehyde dehydrogenase 6 (ALD6) from recombinant Saccharomyces cerevisiae to reduce formaldehyde. The ALD6 gene was cloned under the GAL1 promoter in pYES2 and attached to green fluorescent protein (GFP). To reduce the activity of ALD6, a dominant mutant was constructed with deleted catalytic residues. These strains were successfully transformed in Saccharomyces cerevisiae as confirmed by fluorescence microscopy. The produced enzymes isolated from each strain were used to treat formaldehyde. Formaldehyde reduction was determined via measured luminescence in Vibrio fischeri. Formaldehyde levels were lowest in enzymes from cells overexpressing ALD6. Furthermore, when the strains were exposed to formaldehyde stress, NADH levels increased for strains overexpressing ALD6 and decreased for dominant negative strains. Therefore, our results suggest that ALD6 plays a key role in formaldehyde treatment. We expect that ALD6 could be used in applications related to the removal of formaldehyde.

Effect of GTP-binding protein (YPT1 protein) on the enhanced yeast vacuolar activity.

Yeast GTP-binding protein (YPT1 protein) has been reported to function in the early stages of the secretory pathway. Particularly, YPT1 protein is observed to regulate both the endoplasmic reticulum-to-Golgi transport and the autophagy. Therefore, the YPT1 protein overexpressed in yeast vacuoles is expected to enhance antimicrobial and anticancer activity. The enhancement of yeast vacuolar activity under the overexpression of YPT1 was evaluated by the analysis of lysozyme activity, antimicrobial activity against Escherichia coli and Staphylococcus aureus, and MTT assay against HeLa cell lines. Additionally, the rise in concentration of some important proteinases inside the vacuole, such as proteinase A, proteinase B, and vacuolar carboxypeptidase Y (CPY) were also recorded using a 2DE technique. All results imply YPT1 involvement in the recruitment of some specific proteinases into vacuoles, which leads to the enhancement of vacuolar activity. Since these there proteinases belong to the CPY pathway, YPT1 is even believed to up-regulate this trafficking pathway in yeast cells. Future studies, however, should be carried out to discover the mechanisms that allow YPT1 to recruit these proteins into yeast vacuoles.

Antimicrobial Properties of Lysosomal Enzymes Immobilized on NH2Functionalized Silica-Encapsulated Magnetite Nanoparticles.

The immobilization efficiency, antimicrobial activity and recovery of lysosomal enzymes on NH2 functionalized magnetite nanoparticles have been studied under various conditions. The immobi- lization efficiency depends upon the ratio of the amount of enzyme and magnetite and it shows an increase with magnetite concentration which is due to the presence of amine group at the magnetite surface that leads to a strong attraction. The optimized reaction time to immobilize the lysosomal enzymes on magnetite was determined by using a rolling method. The immobilization efficiency increases with reaction time and reached a plateau after 5 minutes and then remained constant for 10 minutes. However, after 30 minutes the immobilization efficiency decreased to 85%, which is due to the weaker electrostatic interactions between magnetite and detached lysosomal enzymes. The recovery and stability of immobilized lysosomal enzymes has also been studied. The antimicrobial activity was almost 100% but it decreased upon reuse and no activity was observed after its reuse for seven times. The storage stability of lysosomal enzymes as an antimicrobial agent was about 88%, which decreased to 53% after one day and all activity of immobilized lysosomal enzymes was maintained after five days. Thus, the lysosomal enzymes immobilized on magnetite nanoparticles could potentially be used as antimicrobial agents to remove bacteria.

Cellular Lysosomes' Activity for Melanin Reduction on Artificial Skin Tissue.

All eukaryotes have lysosomes which contain hydrolytic enzymes such as protease to degrade waste materials and cellular fragments. As a cellular organelle, lysosomes function as the digestive system of the cell, serving both to degrade material taken up from outside the cell and to digest obsolete components of the cell itself. Conversely, melanin has photochemical functions to protect tissue from the harmful effects of ultraviolet rays. However, too much of melanin leads to problems such as hyperpigmentation, requiring materials to maintain and control the amount of melanin. In this study, we found evidence of correlation between lysosome and melanin in a new eco-friendly material, MelanoDerm, a reconstituted 3D human skin model containing normal melanocytes and keratinocytes. Melanin content assay and cell viability were measured, using 2% kojic acid as positive control, while MelanoDerm was exposed to various concentrations of lysosome. Our results indicate that lysosome may be a useful cosmetic agent for the treatment of hyperpigmentation.

Removal of Trans-2-nonenal Using Hen Egg White Lysosomal-Related Enzymes.

2-Nonenal is a long-chain aliphatic aldehyde containing nine carbons and an unsaturated bond. 2-Nonenal is the primary cause of odor associated with aging, with an unpleasant greasy and grassy odor. Lysosome, mitochondria, and peroxisome are significant organelles in eukaryotic cells that contain various hydrolases that degrade biomolecules. Proteins in mitochondria and peroxisome also contain aldehyde dehydrogenase. We performed trans-2-nonenal treatment using lysosomal-related enzymes extracted from hen egg white (HEW). As trans-2-nonenal is more structurally stable than cis-2-nonenal, it was selected as the target aldehyde. HEW contains various biologically active proteins and materials such as albumin, ovotransferrin, lysosome, peroxisome, and mitochondria. Here, complementary experiments were conducted to evaluate the role of lysosomal-related enzymes in the treatment of trans-2-nonenal. The activity of lysosomal-related enzymes was confirmed via antimicrobial test against E. coli. HPLC analysis was used to determine the reduction of trans-2-nonenal. The trans-2-nonenal treatment depended on the reaction time and enzyme concentration. Materials considered as an intermediate from trans-2-nonenal treatment were detected by GC/MS spectrometer. Under acidic conditions (pH 6), lysosomal-related enzymes were the most efficient in the treatment of trans-2-nonenal. Furthermore, based on differential pH testing, we found the conditions under which all the 50 ppm trans-2-nonenal was removed. Therefore, our results suggest that the lysosomal-related enzymes reduced trans-2-nonenal, suggesting clinical application as anti-aging deodorants.

Treatment of livestock carcasses in soil using Corynebacterium glutamicum and lysosomal application to livestock burial.

A method of rapidly decaying livestock carcasses is sought through Corine glutamicum, and furthermore, lysosomes are used to remove toxic microorganisms from livestock carcasses. The landfill was constructed on a laboratory scale. Optimized growth conditions of C. glutamicum that could quickly decay livestock carcasses were determined. Lysosomes were extracted from egg whites and used to treat contaminated soil to confirm their antimicrobial activities. Condition of C. glutamicum was activated, regardless both anaerobic and aerobic conditions, soil exists and, to be close to the optimum conditions as possible temperatures, moisture content was about 1/10 of the culture. Lysosomes were found to be effective in clearing soil contamination. C. glutamicum can accelerate the decay of livestock carcasses. A combination of C. glutamicum and lysomes could be used to treat soil contamination caused by decomposition of livestock.

Toxicities of Four Parabens and Their Mixtures to Daphnia magna and Aliivibrio fischeri.

The objective of this study was to determine toxicities of four parabens (methyl paraben, MP; ethyl paraben, EP; n-propyl paraben, PP; and n-butyl paraben; BP) and their mixtures to two aquatic microorganisms, Daphnia magna and Aliivibrio fischeri. Parabens are one of the widely used preservatives for personal care products, such as cosmetics, pharmaceuticals and food also. First, each paraben was treated to D. magna to measure the toxicity levels as LC20 and LC50. The results showed their value of MP (25.2 mg/L, 73.4 mg/L), EP (18.4 mg/L, 43.7 mg/L), PP (10.4 mg/L, 21.1 mg/L) and BP (3.3 mg/L, 11.2 mg/L). Then, each of the parabens was treated to A. fischeri and calculated their EC20 and EC50 by bioluminescence inhibition test. The results showed the values of MP (2.93 mg/L, 16.8 mg/L), EP (1.18 mg/L, 6.74 mg/L), PP (0.51 mg/L, 5.85 mg/L) and BP (0.21 mg/L, 2.34 mg/L). These four parabens belong to the group classified as being 'harmful to aquatic organisms' (above 10 mg/L, below 100 mg/L). After measuring the toxicity, EC20 values of two or more parabens were tested in order to investigate their toxicity. A total of ten combinations of four parabens were tested. As a result, the bioluminescence inhibition test of A. fischeri showed that the toxicity of mixture parabens was stronger than that of a single compound and combinations of three parabens showed the highest bioluminescence inhibition. These results showed that independent toxicity of paraben was maintained. Therefore, it can be predictable that the toxicity of paraben is getting stronger by the addition of other parabens.

The Response of Liposomes Modified with Lysosomal Membrane Proteins to the Targeting Ability Associated with Antimicrobial Activity.

Liposomes were modified using two different methods. In Method 1, liposomes were modified by mixing whole lysosomal proteins, lipid, and cholesterol before preparation. For Method 2, the liposomes were modified by mixing whole lysosomal proteins after liposome preparation. Method 1-modified liposomes exhibited improved cell mortality compared to Method 2-modified liposomes. The modified liposomes were then evaluated for their antimicrobial activity against lysosomal enzymes, and Escherichia coli did not modify the liposome surface. The whole lysosomal membrane proteins extracted from the lysosomes in Saccharomyces cerevisiae were analyzed using two-dimensional electrophoresis to find specific proteins associated with antimicrobial activity and to construct recombinant S. cerevisiae proteins. Additionally, genes related to antimicrobial activity were identified, and the liposomes modified by lysosomal membrane proteins of recombinant S. cerevisiae tagged with green fluorescence proteins were prepared and overexpressed. The modified liposomes exhibited improved antimicrobial activity with an almost two-fold increase in the cell mortality rate, suggesting crucial roles as potential therapeutics.

Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells.

OBJECTIVES: There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. METHODS: It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. RESULTS: The result showed 14 up-regulated (1.5-fold) and 13 down-regulated (2.0-fold) spots in relation to melanin exposure. CONCLUSIONS: It has been found that lysosomal membrane proteins are associated with melanin to decolorize and quantity through cellular activation of lysosome.

Outstanding Antibiofilm Features of Quanta-CuO Film on Glass Surface.

Intelligently designed surface nanoarchitecture provides defined control over the behavior of cells and biomolecules at the solid-liquid interface. In this study, CuO quantum dots (quanta-CuO; ∼3-5 nm) were synthesized by a simple, low-temperature solution process and further formulated as paint to construct quanta-CuO thin film on glass. Surface morphological characterizations of the as-coated glass surface reveal a uniform film thickness (∼120 ± 10 nm) with homogeneous distribution of quanta-CuO. The antibiofilm assay showed a very high contact bacteria-killing capacity of as-coated quanta-CuO glass surfaces toward Staphylococcus aureus and Escherichia coli. This efficient antibacterial/antibiofilm activity was ascribed to the intracellular reactive oxygen species (ROS) generated by the quanta-CuO attached to the bacterial cells, which leads to an oxidative assault and finally results in bacterial cell death. Although there is a significant debate regarding the CuO nanostructure's antibacterial mode of action, we propose both contact killing and/or copper ion release killing mechanisms for the antibiofilm activity of quanta-CuO paint. Moreover, synergism of quanta-CuO with conventional antibiotics was also found to further enhance the antibacterial efficacy of commonly used antibiotics. Collectively, this state-of-the-art design of quanta-CuO coated glass can be envisioned as promising candidates for various biomedical and environmental device coatings.

Effect of acid trehalase (ATH) on impaired yeast vacuolar activity.

In this study, this protein was overexpressed in yeast cells grown on trehalose-containing medium to assess its impact on yeast vacuolar activity. ATH was confirmed to be located in both cell surface and vacuoles and the overexpression of ATH was observed to decrease vacuolar activity. Therefore, an assumption was suggested to explain this phenomenon as follows: when grown on containing trehalose medium, the ATH localization at cellular periplasm, but not the vacuole, is prioritized to utilize the extracellular trehalose for cell growth. The multivesicular body pathway (MVB pathway) via which ATH is transported into vacuoles is believed to be down-regulated to favor the accumulation of ATH at cell surface area. By extension, other vacuolar proteins travelling through MVB pathway to reach yeast vacuoles likely also suffer the down regulation. It can be concluded that acid trehalase may contribute down regulation of other vacuolar proteins through MVB pathway. This study suggests that it is a potential of acid trehalase (ATH) on impaired activity of yeast vacuolar.

Impact of Solvent pH on Direct Immobilization of Lysosome-Related Cell Organelle Extracts on TiO2 for Melanin Treatment.

Techniques for immobilizing effective enzymes on nanoparticles for stabilization of the activity of free enzymes have been developing as a pharmaceutical field. In this study, we examined the effect of three different pH conditions of phosphate buffer, as a dissolving solvent for lysosomal enzymes, on the direct immobilization of lysosomal enzymes extracted from Hen's egg white and Saccharomyces cerevisiae. Titanium(IV) oxide (TiO2) nanoparticles, which are extensively used in many research fields, were used in this study. The lysosomal enzymes immobilized on TiO2 under each pH condition were evaluated to maintain the specific activity of lysosomal enzymes, so that we can determine the degree of melanin treatment in lysosomal enzymes immobilized on TiO2. We found that the immobilization efficiency and melanin treatment activity in both lysosomal enzymes extracted from Hen's egg white and S. cerevisiae were the highest in an acidic condition of phosphate buffer (pH 4). However, the immobilization efficiency and melanin treatment activity were inversely proportional to the increase in pH under alkaline conditions. In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca(2+), and the melanin treatment activity of immobilized lysosomal enzymes on TiO2 pretreated with Ca(2+) was also increased. Therefore, this result suggests that the immobilization efficiency and melanin treatment activity of lysosomal enzymes can be enhanced according to the pH conditions of the dissolving solvent.

Tailored lysozyme-ZnO nanoparticle conjugates as nanoantibiotics.

Covalently attached lysozyme-ZnO nanoparticle (L-ZNP) conjugates were synthesized by a low temperature solution route. Tailored L-ZNP conjugates exhibit pronounced antibacterial features against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).

Toxic detection in mine water based on proteomic analysis of lysosomal enzymes in Saccharomyces cerevisiae.

OBJECTIVES: Lysosome is the cell-organelle which is commonly used as biomonitoring tool in environmental pollution. In this study, the lysosomal proteomic of the yeast Saccharomyces cerevisiae was analyzed for utilization in the detection of toxic substances in mine water samples. METHODS: This work informs the expression of lysosomal proteomic in yeast in response with toxic chemicals, such as sodium meta-arsenite and tetracycline, for screening specific biomarkers. After that, a recombinant yeast contained this biomarker were constructed for toxic detection in pure toxic chemicals and mine water samples. RESULTS: Each chemical had an optimal dose at which the fluorescent protein intensity reached the peak. In the case of water samples, the yeast showed the response with sample 1, 3, 4, and 5; whereas there is no response with sample 2, 6, and 7. CONCLUSIONS: The recombinant yeast showed a high ability of toxic detection in response with several chemicals such as heavy metals and pharmaceuticals. In the case of mine water samples, the response varied depending on the sample content.

Phenol degradation activity and reusability of Corynebacterium glutamicum coated with NH(2)-functionalized silica-encapsulated Fe3O4 nanoparticles.

In this study, a novel method to immobilize and separate Corynebacterium glutamicum for phenol degradation was developed using Fe(3)O(4) nanoparticles (NPs). The Fe(3)O(4) NPs were encapsulated with silica and functionalized with NH(2) groups to enhance their capacity to adsorb on the cell surface. The results showed that the NH(2)-functionalized silica-encapsulated Fe(3)O(4) NPs strongly adsorbed on the cell surface of C. glutamicum during 32 d culture without any interruptions of their normal cell growth. The coated C. glutamicum were easily separated from the culture broth within 2 min by applying an external magnetic field Also, the coated C.glutamicum were able to completely degrade 50 ppm phenol in the culture broth after 8d culture at 30 °C. Concerning reusability, the coated cells could completely degrade phenol during the first 2 cycles, and retain ~60% activity of phenol degradation for the third and four cycles.

Increased in vitro lysosomal function in oxidative stress-induced cell lines.

Exposure of mammalian cells to oxidative stress alters lysosomal enzymes. Through cytochemical analysis of lysosomes with LysoTracker, we demonstrated that the number and fluorescent intensity of lysosome-like organelles in HeLa cells increased with exposure to hydrogen peroxide (H2O2), 6-hydroxydopamine (6-OHDA), and UVB irradiation. The lysosomes isolated from HeLa cells exposed to three oxidative stressors showed the enhanced antimicrobial activity against Escherichia coli. Further, when lysosomes that were isolated from HeLa cells exposed by oxidative stress were treated to normal HeLa cells, the viability of the HeLa cells was drastically reduced, suggesting increased in vitro lysosomal function (i.e., antimicrobial activity, apoptotic cell death). In addition, we also found that cathepsin B and D were implicated in increased in vitro lysosomal function when isolated from HeLa cells exposed by oxidative stress. Decrease in cathepsin B activity and increase in cathepsin D activity were observed in lysosomes isolated from HeLa cells after treatment with H2O2, 6-ODHA, or UVB, but cathepsin B and D were not the sole factors to induce cell death by in vitro lysosomal function. Therefore, these studies suggest a new approach to use lysosomes as antimicrobial agents and as new materials for treating cancer cell lines.

Evaluation of whole lysosomal enzymes directly immobilized on titanium (IV) oxide used in the development of antimicrobial agents.

Lysosomal enzymes isolated from egg white were directly immobilized on titanium (IV) oxide (TiO(2)) particles using shaking methods (150 rpm, room temperature, 10 min), and the immobilization efficiency, activity, and stability of lysosomal enzymes immobilized on TiO(2) were evaluated. Of the various mass ratios (w/w) of lysosomal enzymes to TiO(2) tested, we found that 100% immobilization efficiency was observed at a ratio of 1:20 (enzymes:TiO(2); w/w). Furthermore, the antimicrobial activities of the immobilized lysosomal enzymes were confirmed using viable cell counts against Escherichia coli. Our results showed that the antimicrobial activity of immobilized lysosomal enzymes is stable and can be maintained up to one month, but the antimicrobial activity of free enzymes without immobilization completely disappeared after five days in storage. In addition, enhanced immobilization efficiency was shown in TiO(2) pretreated with a divalent, positively charged ion, Ca(2+), and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized enzymes. However, K(+), a monovalent, positively charged ion, did not have any positive effect on immobilization or antimicrobial activity. Finally, we suggest that activity and stability of immobilized lysosomal enzymes can be maintained for a longer time than those properties of free lysosomal enzymes.

Toxicity Assessment of Titanium (IV) Oxide Nanoparticles Using Daphnia magna (Water Flea).

OBJECTIVES: Titanium dioxide (TiO(2)), a common nanoparticle widely used in industrial production, is one of nano-sized materials. The purpose of this study was to determine the acute and chronic toxicity of TiO(2) using different size and various concentrations on Daphnia magna. METHODS: In the acute toxicity test, four concentrations (0, 0.5, 4, and 8 mM) for TiO(2) with 250 or 500 nm and five concentrations (0, 0.25, 0.5, 0.75, and 1 mM) for TiO(2) with 21 nm were selected to analyze the toxic effect to three groups of ten daphnia neonates over 96 hours. In addition, to better understand their toxicity, chronic toxicity was examined over 21 days using 0, 1, and 10 mM for each type of TiO(2). RESULTS: Our results showed that all organisms died before the reproduction time at a concentration of 10 mM of TiO(2). In addition, the exposure of anatase (21 nm) particles were more toxic to D. magna, comparing with that of anatase (250 nm) and rutile (500 nm) particles. CONCLUSIONS: This study indicated that TiO(2) had adverse impacts on the survival, growth and reproduction of D. magna after the 21days exposure. In addition, the number of test organisms that were able to reproduce neonates gradually were reduced as the size of TiO(2) tested was decreased.