Yong-Keun Choi

Ionic Liquid-mediated Extraction of Lipids from Algal Biomass

Lipids from algal biomass were extracted using mixtures of ionic liquids (ILs) and methanol, and fatty acid profiles of the extracted lipids were characterized in this work. Mixtures of ILs and methanol successfully dissolved biomass leaving lipids insoluble. The total contents of lipids extracted from commercial and cultivated Chlorella vulgaris were 10.6% and 11.1%, respectively, by the conventional Bligh and Dyers method, while a mixture of [Bmim][CF(3)SO(3)] and methanol extracted 12.5% and 19.0% of the lipids, respectively. Multi-parameter regression by the linear solvation energy relationship showed that dipolarity/polarizability and hydrogen bond acidity of ILs are more important than their hydrogen bond basicity for effectively extracting lipids from algal biomass. Fatty acid profiles of the lipids extracted using IL-methanol mixtures showed that C16:0, C16:1, C18:2, and C18:3 fatty acids were dominant. This suggests that the lipids extracted from C. vulgaris can be used as a source of biodiesel production.

Isolation of taxol, an anticancer drug produced by the endophytic fungus, Phoma betae

Phoma betae, an endophytic fungus, was isolated from the healthy leaves of Ginkgo biloba . The fungus was screened for the production of taxol on a modified liquid medium for the first time. The fungal species were identified by their characteristic culture morphology and molecular analysis. The presence of taxol was confirmed by spectroscopic: ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), liquid chromatography–mass spectrometry (LC-MS); and chromatographic: thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods of analysis. The taxol production was quantified by HPLC analysis. The maximum amount of taxol production was recorded as 795 μg/L. The production rate was 15,900-fold more than that found in the culture broth of earlier reported fungus, Taxomyces andreanae . The extracted fungal taxol demonstrate a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay. This indicates that the increase in taxol concentration induced an increased cell death. A polymerase chain reaction (PCR) based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton was confirmed as a molecular blueprint for taxol biosynthesis. These results designate that the fungus, P. betae is an excellent candidate for taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level. Key words : Taxol production, Phoma betae, analytical methods, cytotoxicity assay, taxadiene synthase.

LED light stress induced biomass and fatty acid production in microalgal biosystem, Acutodesmus obliquus.

Microbial algal system can serve as a potential source for the production of much high value bioproducts and biofuels. The quality and intensity of light are the key elements to optimize the production of algal biomass and fatty acid contents. This study presents the effect of differential LED flashing light conditions on the growth of microalgae, Acutodesmus obliquus. The induced light stress was optimized for its biomass and fatty acid content. The microalgae are exposed to various frequency of intermittent LED flashing light (blue and red lights) at three different phases in the 18 day cell growth (log, lag and stationary phase). The frequency of light flashing rate was adjusted to 120, 10, 5, 3.75, and 1 times per min. The effect of light stress on growth and fatty acids composition of A. obliquus induced an increase in algae growth and fatty acid production. Different optimal timing for light stress was subjected to elucidate the effect of light stress on algae growth and fatty acid production. The results showed an increase in the algae growth (1.2mg/L of chl a content) under light stress condition at FT10 (flashing time, 10 times per min) from the initial day (log phase) compared with the control experiment (0.4 mg/L of chl a content). However, the total fatty acids (71 mg/g) and volumetric FAME production (9.4 ml/l) level was found to be significant under FT5 (flashing time, 5 times per min), adopting flashing light from day 10 (stationary phase). TEM studies also revealed the deposition of lipid to be largest in the 18 day old cells under flashing light (FT5) condition, representing maximum accumulation of lipids bodies (up to 770 nm diameter in particle size) occupying approximately 42% of the total area of the cell.

In Vitro Cytotoxic Evaluation of MgO Nanoparticles and Their Effect on the Expression of ROS Genes

Water-dispersible MgO nanoparticles were tested to investigate their cytotoxic effects on oxidative stress gene expression. In this in vitro study, genes related to reactive oxygen species (ROS), glutathione S-transferase (GST) and catalase, were quantified using real-time polymerase chain reactions (molecular level) and molecular beacon technologies (cellular level). The monodispersed MgO nanoparticles, 20 nm in size, were used to treat human cancer cell lines (liver cancer epithelial cells) at different concentrations (25, 75 and 150 µg/mL) and incubation times (24, 48 and 72 h). Both the genetic and cellular cytotoxic screening methods produced consistent results, showing that GST and catalase ROS gene expression was maximized at 150 µg/mL nanoparticle treatment with 48 h incubation. However, the genotoxic effect of MgO nanoparticles was not significant compared with control experiments, which indicates its significant potential applications in nanomedicine as a diagnostic and therapeutic tool.

Glucose oxidase/cellulose-carbon nanotube composite paper as a biocompatible bioelectrode for biofuel cells.

Biofuel cells are devices for generating electrical energy directly from chemical energy of renewable biomass using biocatalysts such as enzymes. Efficient electrical communication between redox enzymes and electrodes is essential for enzymatic biofuel cells. Carbon nanotubes (CNTs) have been recognized as ideal electrode materials because of their high electrical conductivity, large surface area, and inertness. Electrodes consisting entirely of CNTs, which are known as CNT paper, have high surface areas but are typically weak in mechanical strength. In this study, cellulose (CL)–CNT composite paper was fabricated as electrodes for enzymatic biofuel cells. This composite electrode was prepared by vacuum filtration of CNTs followed by reconstitution of cellulose dissolved in ionic liquid, 1-ethyl-3-methylimidazolium acetate. Glucose oxidase (GOx), which is a redox enzyme capable of oxidizing glucose as a renewable fuel using oxygen, was immobilized on the CL–CNT composite paper. Cyclic voltammograms revealed that the GOx/CL–CNT paper electrode showed a pair of well-defined peaks, which agreed well with that of FAD/FADH2, the redox center of GOx. This result clearly shows that the direct electron transfer (DET) between the GOx and the composite electrode was achieved. However, this DET was dependent on the type of CNTs. It was also found that the GOx immobilized on the composite electrode retained catalytic activity for the oxidation of glucose.

Long-term follow-up of porcine male germ cells transplanted into mouse testes.

This study investigated the effect of increased phylogenetic distance on the outcome of spermatogonial transplantation, with porcine donors and mice recipients. It was designed to develop a technique for detecting foreign donor cells in recipient animals. Porcine male germ cells were harvested from postnatal male testes and incubated with the lipophilic membrane dye PKH-26. For transplantation, approximately 10(6) PKH-26-labelled porcine male germ cells were injected into the efferent ducts of mouse testes. Animals were sacrificed at post-graft days 1, 10, 30, 45, 60 and 150 (n = 5 each). Serial frozen sections of explanted testes were prepared for detecting labelled cells. Transplanted porcine donor cells were easily detected in the recipient tubules for 8 weeks. After transplantation, we could detect both incorporation into the basement membrane and differentiation of grafted porcine donor cells by our double detection system, using PKH staining and slide PCR. However, our RT-PCR and apoptosis results revealed that most of the grafted porcine male donor cells could not differentiate past early-meiotic spermatocytes. We could induce partial differentiation of xenografted porcine donor cells in mouse testes, but not full induction of spermatogenesis. We have developed a very reliable technique for detecting foreign donor cells in recipient animals using a combination of PKH staining and slide PCR methods. Our results provide a valuable experimental model for applying and evaluating this technology in other species.

Enhanced growth and total fatty acid production of microalgae under various lighting conditions induced by flashing light

Microalgae are gaining importance as a source of high‐value bioproducts. However, data regarding optimization of algal productivity via variation of environmental factors are lacking. Here, we evaluated a novel lighting method for the enhancement of biomass and total fatty acid (TFA) productivities during algal cultivation. We cultivated six different algal strains (Chlorella vulgaris KCTC AG10002, Acutodesmus obliquus KGE18, Uronema sp. KGE03, Micractinium reisseri KGE19, Fragilaria sp., and Spirogyra sp.) under various lighting conditions—continuous light (CL), light‐dark cycle (LD), and continuous dark (CD)—with or without additional flashing light. We monitored dry cell weight (DCW) and TFA concentrations during cultivation. For each algal strain, the growth rate showed markedly different responses to the various lighting modes. The growth rates of C. vulgaris KCTC AG10002 (1.34‐fold DCW increase, LD with flash), A. obliquus KGE18 (5.16‐fold DCW increase, LD with flash), Uronema sp. KGE03 (2.77‐fold DCW increase, CL with flash), and M. reisseri KGE19 (1.52‐fold DCW increase, CL with flash) markedly increased in response to flashing light. Additionally, in some algal strains cultivated under the LD mode, the flashing light treatment induced increased TFA concentrations (C. vulgaris, 1.19‐fold increase; A. obliquus, 2.59‐fold increase; and M. reisseri, 3.31‐fold increase). Phytohormone analysis of M. reisseri revealed increases in growth rate and TFA concentrations, associated with phytohormone induction via flashing light (e.g. 2.93‐fold increase in gibberellic acid); hence, flashing light can promote substantial alterations in algal metabolism.

403 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL MOUSE AND HUMAN MOPT GENE CONTAINING MORN MOTIF PROTEIN IN TESTIS

By subtraction screening methods, we identified a novel mouse and its counterpart, human MOPT gene. The mouse and human MOT gene is localized on mouse chromosome 17E3 and human chromosome 2p22, and spans approximately 7 kb. Analysis of the mouse Mopt sequence revealed the existence of an ORF of 240 bp encoding a putative protein of 79aa amino acids. The predicted protein has a theoretical molecular mass of 8.89 kDa and a calculated isoelectric point of 5.82. The protein was unique; it did not show any similarities with other known protein except for Morn motif domain. Real-time reverse transcriptase polymerase chain reaction and Northern blot analysis revealed that mouse Mopt transcripts are highly and specifically expressed in adult testis and skeletal muscle. In situ hybridization and immunohistochemistry studies showed that mouse Mopt transcript and protein was confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increases in abundance in subsequent stages. To characterize Mopt functions in spermiogenesis, we examined Mopt protein distribution in late spermiogenesis by using immunogold electron microscopy: Mopt protein first appeared in the proacrosomic vesicles of the early Golgi phase spermatids. In the final step of spermiogenesis, Mopt expression was translocated from the head cap of an elongated spermatid to the nucleus of mature spermatozoa. However, no other testicular cell types, including somatic cells, spermatogenic cells, and residual cytoplasm that ultimately is engulfed as residual bodies into Sertoli cells, were found to bear Mopt-positive staining. This observation suggested that Mopt may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis and an as yet uncharacterized role in oocyte activation during capacitation, after fertilization, or both.

263 IDENTIFICATION OF A NOVEL MOPT GENE IN HUMAN AND MOUSE ADULT TESTIS

To discover late stage germ cell-specific transcripts we prepared a cDNA library from adult testes of 35-day old mice and subtracted it with mRNA from the testes of juvenile mice. Real-time RT-PCR analysis indicated that 42 cDNA clones in the subtracted library were expressed more intensely in the adult testes than in the juvenile testes. One clone identified by subtraction is expressed preferentially in the late spermatid and is located on chromosome 17E3 in mouse and 2p22 in human. The full nucleotide and amino acid sequences of mouse and human MOPT gene are deposited in EMBL GenBank (AY367765 And AY367766). Human MOPT is spliced by 5 exons and 4 introns and encompasses 7,000 bp of genomic DNA (from bp 355 822 to 425 511) of NT-022184.13, whereas mouse MOPT is spliced by 5 exons and 4 introns and encompasses 7,382 bp of genomic DNA (from bp 6227407 to 6235588) of NT-039658.2. Because of the limited availability of human testis samples, development-dependent expression of MOPT mRNA was conducted using its mouse homologue and semiquantitative PCR. The number of cycles completed before entering the exponential growth, recorded by amplifier PE5700 for mouse MOPT, were 1.11 ± 0.23, 1.05 ± 0.04, 1.5 ± 0.2, 5.55 ± 0.65, 19.35 ± 0.65, 68.65 ± 2.15, and 185.15 ± 6.15 in W/W, postnatal day 5-, 8-, 12-, 15-, 18-, 22-, and 28-day mouse tissue samples, respectively. The difference among the three times was significant (P < 0.01, ANOVA). These results suggest that expression of MOPT gene increased from postnatal Day 5 to Day 28, indicating possible involvement in testicular development. The ORF encodes a protein containing 79 amino acid residues. A MORN motif, EGQFKDNMFHGLGTYTFPNG, was identified in the predicted protein sequence of MOPT; function of this motif is unknown. In situ hybridization of 12-week-old wild-type mouse testes using an antisense riboprobe and immuno-gold data indicated MOPT was expressed as a late spermatid and acrosome reaction. This work was supported by BK21 program.