Jihee Yoon

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.

Melanin reduction by peroxidase activity in lysosome-related organelle extracts from hen egg whites, HeLa cells, and Saccharomyces cerevisiae

Although melanin plays a biological role in the protection of underlying tissues from harmful ultraviolet radiations, yet large amounts of melanin in light-skinned individuals can make them susceptible to skin cancer. In this study, lysosome-related organelles extracts (LOE) from hen egg whites, HeLa cells, and Saccharomyces cerevisiae have been used to lower melanin color intensity. The results show 80% reduction in melanin upon daily treatment with 100 μg LOE for a period of 19 days. Additionally, the treatment with S. cerevisiae LOE showed the best melanin color reduction, which is consistent with the level of peroxidase activity. The treatment of LOE from S. cerevisiae with NH4Cl decreases melanin reduction activity and peroxidase activity compare to LOE from normal S. cerevisiae. Meanwhile, in contrast, the treatment with H2O2 showed a completely contrary pattern. The above results thus indicate that LOEs can be promising agents for use in cosmeceutical skin lightening as well as for the treatment of hyper pigmentation disorders.

Biodegradation of diisodecyl phthalate (DIDP) by Bacillus sp. SB-007.

In this study, diisodecyl phthalate (DIDP) was efficiently degraded by Bacillus sp. SB‐007. The optimal conditions for DIDP (100 mg l–1) degradation by Bacillus sp. SB‐007 in a mineral salts medium were found to be pH 7.0 at 30 °C, stirring at 200 rpm. The specific rate of DIDP degradation was found to be concentration dependent with a maximum of 4.87 mg DIDP l–1 h–1. DIDP was transformed rapidly by Bacillus sp. SB‐007 with the formation of monoisodecyl phthalate and phthalic acid, which subsequently degraded further. These results highlight the potential of this bacterium for removing DIDP contaminated waste in the environment. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Differential Proteomic Analysis of Secreted Proteins from Cutinase-producing Bacillus sp. SB-007

Bacillus sp. SB-007 was isolated from pea leaves harvested from the southwestern parts of South Korea through screening on a minimal medium containing 0.2% purified cutin for its ability to induce the cutinase production. However, no cutinase was produced when it was grown in a minimal medium containing 0.2% glucose. A proteomic approach was applied to separate and characterize these differentially secreted proteins. The expression level of 83 extracellular proteins of the cutinase-producing Bacillus sp. strain SB-007 incubated in a cutinase-induced medium increased significantly as compared with that cultured in a non cutinase-induced medium containing glucose. The extracellular proteome of Bacillus sp. SB-007 includes proteins from different functional classes, such as enzymes for the degradation of various macromolecules, proteins involved in energy metabolism, sporulation, transport/binding proteins and lipoproteins, stress inducible proteins, several cellular molecule biosynthetic pathways and catabolism, and some proteins with an as yet unknown function. In addition, the two protein spots showed little similarities with the known lipolytic enzymes in the database. These secreted proteome analysis results are expected to be useful in improving the Bacillus strains for the production of industrial cutinases.

Color reduction of melanin by lysosomal and peroxisomal enzymes isolated from mammalian cells

Lysosomes and peroxisomes are organelles with many functions in all eukaryotic cells. Lysosomes contain hydrolytic enzymes (lysozyme) that degrade molecules, whereas peroxisomes contain enzymes such as catalase that convert hydrogen peroxide (H2O2) to water and oxygen and neutralize toxicity. In contrast, melanin is known as a helpful element to protect the skin against harmful ultraviolet rays. However, a high quantity of melanin leads to hyperpigmentation or skin cancer in human. New materials have already been discovered to inhibit tyrosinase in melanogenesis; however, melanin reduction does not suggest their preparation. In this study, we report that the color intensity because of melanin decreased by the cellular activation of lysosomes and peroxisomes. An increase in the superficial intensity of lysosome and peroxisome activities of HeLa cells was observed. In addition, a decrease in the amount of melanin has also been observed in mammalian cells without using any other chemical, showing that the process can work in vivo for treating melanin. Therefore, the results of this study indicate that the amount of melanin decreases by the lysosome and peroxisome activity after entering the cells, and functional organelles are effective in color reduction. This mechanism can be used in vivo for treating melanin.

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.

Lysosome-related organelles extracts from egg-white for hypermelanosis reduction

Pigmentary disorders generally indicate that an increased amount of melanin, leading to darker color of skin, called hypermelanosis. Thus, several methods were studied for finding therapy of hypermelanosis until now in worldwide. In this study, the hypermelanosis reduction has been observed with daily lysosome-related organelles (LRO) extract from eggwhite. The scrub was used for enzyme extraction so as to maintain the stability of the extracted enzyme. The LRO extracts were divided and into two groups: free enzyme and fresh enzyme. Free enzyme was extracted on the first day from the LRO and fresh enzyme was extracted by scrub from LRO daily. The melanin dissolved phosphate buffer (pH 7.0) was treated by two groups of enzymes for a period of 30 days. The fresh enzymes were found to be more effective than the free enzymes. The activity of free enzymes decreases with time and as a result it could not degrade melanin after 36 days. The peroxidase activity was also measured and it matched with the results of previous studies. Therefore, the daily fresh enzymes displayed remarkable ability in hypermelanosis reduction than with free enzyme that lost its activity after some time. The present work can be potentially used for dermal disease in medical industry and also in cosmetic products.

Proteomic analysis of Daphnia magna exposed to glyphosate and methidathion

Proteomic analysis was performed to identify proteins involved in the stress responses of Daphnia magna to glyphosate and methidathion. Lethal concentration of chemicals causing 50% mortality (LC50) determined by acute toxicity assays were used to expose to daphnias from 24 h to 21 days prior to total protein extraction. The proteomic profile of tested organisms was analyzed by the two-dimensional electrophoresis method. The average intensity of each spots was then analyzed to determine the differently expressed proteins (DEPs). The DEPs was considered as down- or up-regulations only if their expression level in control samples are linearly higher than LC50 or lower than LC50, respectively. The result showed that there are 22 and 27 DEPs responding to the exposure of glyphosate and methidathion, respectively. The expression pattern of established DEPs is helpful to understand the molecular response of D. magna to glyphosate and methidathion, and discover potential candidates as novel biomarkers.

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.