Earticle

Miscanthus is one of major crop for bio-energy, its biomass is usually more than those of other crops and culture condition is suitable for Korean climate. Miscanthus’s potential biomass will be 42ton / ha, which around tow times more than one of switchgrass. In this study, we were focused on optimal condition of pretreatment for lignocellulosic biomass in miscanthus by using ammonia. Materials were analyzed to NREL standard quantification of cellulosic biomass. Miscanthus consists of 35~42% of cellulose, 22~25% of hemicellulose and 23~25% of lignin. To find of optimal conditions for effective fractionation of carbohydrates and their conversion process into sugars were checked; hydrolyzing the sample with 10~20% aqueous ammonia at 150~190 for 20~60 minutes. For effective hydrolysis, an oilbath with high temperature control was used and sample was hydrolyzed by using 15mL cylinder type reactor. The best concentration of samples for the reaction was 7.5%. To improve of ethanol conversion ratio, economical and effective pretreatment conditions have to be developed such as combination of aqueous ammonia and diluted acid treatment. Fermentable concentration of sample and optimal culture medium should be considered.

Eichhornia crassipes is macrophyte that originated in Amazon in South America. Eichhornia crassipes is utilized for purification of water or as a feedstock of producing useful materials and feed of animals. Eichhornia crassipes is one of the potential biomass for production of bioethanol, because of it grows fast and it is not edible [1]. For the production of bioethanol, most effective pretreatment method of water hyacinth is alkaline/oxidative pretreatment (A/O pretreatment) using NaOH/H2O2 solution [2]. In this study, to increase the production of bioethanol we optimized A/O pretreatment condition and hydrolysis condition. For hydrolysis of biomass, different commercial enzymes were used. A/O pretreatment was treated in different NaOH/H2O2 concentration and reaction temperature. Enzymatic hydrolysis was performed under different enzyme concentrations and enzyme ratios. After ethanol fermentation using Saccharomyces cerevisiae KCTC 7928, the concentration of glucose and ethanol was analyzed by HPLC with RI.

In this study, the feasibility of using vegetable oil extracted from tropical crop seed as a biodiesel feedstock was investigated by producing biodiesel and analyzing the quality parameters as a transport fuel. In order to produce biodiesel efficiently, two step reaction processes (pre-treatment and transesterification) were required because the tropical crop oil have a high content of free fatty acids.To determine the suitable acid catalyst for the pre-esterification, three kinds of acid catalysts were tested and sulfuric acid was identified as the best catalyst. After constructing the experiment matrix based on RSM and analyzing the statistical data, the optimal pre-treatment conditions were determined to be 26.7% of methanol and 0.982% of sulfuric acid. Trans-esterification experiments of the pre-esterified oil were carried out. As a result, the optimal trans-esterification conditions were 0.8% of KOH and 16.13% of methanol. After trans-esterification of tropical crop oil, the produced biodiesel could meet the major quality standard specifications; 100.8% of FAME, 0.45 mgKOH/g of acid value, 0.00% of water, 0.04% of total glycerol, 4.041 mm2/s of dynamic viscosity at 40 ℃.

Barley straw is a lignocellulosic biomass that is a renewable organic substance and alternative source of energy. In this study, barley straw was converted to ethanol by Saccharomyces cerevisiae following dilute acid pretreatment. barley straw was treated at various sulfuric acid concentrations and reaction temperatures and times. Barely straw Optimize pretreated condition was in 1% dilute acid at 158℃ for between 12min. The pretreated samples were subjected to enzymatic digestibility test by using commercial enzymes. Following enzyme hydrolysis, Saccharomyces cerevisiae was inoculated for ethanol production. The structural change of barley straw after pretreatment and enzyme hydrolysis was examined by SEM and XRD. The results were analyzed to assess the overall effectiveness of the pretreatment.

Ethanol production processes using crops have been well established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel, without the ethical concerns associated with the use of potential food resources. This study, in this sense, dealt with the diluted acid pretreatment of cassava stem as lignocellulosic biomass, following by enzymatic saccharification and fermentation to form ethanol by S. cerevisiae. Pretreatment conditions were optimized using response surface methodology (RSM) allowing the study on influence of the different variables (temperature, time, acid concentration). As a result, the optimal conditions were established for the pretreatment process with temperature at 177°C, 10 min of reaction time and acid concentration of 0.14M. In case of saccharification, enzymatic digestibility was fixed at approximately 70% with cellulase 20 FPU/g cellulose considering economic feasibility. Also, ethanol concentration and yield were attained at 7.55 g/L in 24 h and 89.6%, respectively. This study presented not only the overall process but also an efficient bioethanol production from lignocellulosic materials.

Sea algae consist mainly of 40~70% polysaccharides, 10~20% proteins, and residual low-molecular-weight compounds such as fatty acids, free amino acids, and amines1. While polysaccharides of sea algae such as algine, agar, and carageenin have been used traditionally as food additive, sea algae cellulose has been little utilized. In sea algae, the content of cellulose is less than the water-soluble polysaccharides and highly crystallined structure of cellulose makes the cellulase-catalyzed hydrolysis reaction in aqueous media slow2-3. Since the pretreatment process of sea algae plays an important role in effective conversion of sea algae polysaccharides to glucose by enzymatic hydrolysis. Microwave irradiation was used, in this study, for the pretreatment of sea algae, Ulva pertusa Kjellman. The effect of pretreatment conditions on enzymatic hydrolysis of sea algae were investigated.

The depletion of petroleum-based fuels and environmental problems has stimulated a development of inexpensive biofuels production. The lignocellulosic materials have been focused as the economically attractive substrate for the production of biofuels. Lignocellulose is abundant and renewable resources with great potential as substrates for biofuel fermentation. Hardwood hemicelluloses contain mostly xylan that 60-70% of its residues are acetylated, which leads to the formation of acetic acid by peeling off during the hydrolysis process. The acetic acid in lignocellulose hydrolysate inhibits seriously the cell growth and ethanol production by microorganisms. In this study, alkaline-pretreatment method was introduced for the extraction of acetic acid from xylan of hemicellulose, prior to concentrated acid hydrolysis of yellow poplar wood meal. Ethanol fermentability in deacetylated yellow poplar hydrolysate (DYPH) by Pichia stipitis was also investigated. The alkali-pretreatment conditions were elucidated in terms of temperature, reaction time, and alkalinity. The 94% of acetyl group in xylan of yellow poplar hemicellulose fraction was extracted by 0.5% sodium hydroxide solution at 60 ℃ during 60 min. P. stipitis was affected seriously on cell growth and ethanol production in the synthetic medium added 7.1 g/l of acetic acid or yellow poplar hydrolysate (YPH) containing 7.1 g/l of acetic acid. On the other hand, ethanol production in DYPH was slightly higher than that of the control although cell growth decreased by 34%. The alkaline-pretreatment method greatly enhanced the ethanol fermentability of yellow poplar hydrolysate.

In this study, the fermentation conditions of the repeated batch process were investigated for the commercialization of bioethanol production from corn (Gangdaok) as domestic material. Also, the value as feedstuff of Gangdaok fermentation by-product was evaluated in comparison with USA corn (Ambrosia), which is a widely used feedstock. In order to carry out the repeated batch process of Gandaok, various fermentation conditions were examined. With the purified glucoamylase using and 1 g/L urea supplement, a series of the repeated batch operations was successfully carried out. The final ethanol concentration, the ethanol yield, and the volumetric productivity were attained to 85 g/L, 90%, and 1.77 g/L‧h, respectively. Furthermore, the DDGS of Gangdaok contained more essential amino acids (21.1 mg/g) than Ambrosia. The results showed that Gangdaok has potential economic advantages if applied to the bioethanol and feed industries.

The pesticide activities of biotic pesticide [Sophora flavescens Aiton (30%), Derris elliptica (30%) , Neem oil (30%) extrct] was tested by the broth dilution method and leaf disk method against Tetranichus Urticae, Myzus persicae. The Biotic pesticide was observed 1day, 3day, 5day. The result of using biotic pesticide extracts as a pesticide showed over 89.5%, 73.1%, 70.1% succes rate within 5 day in eliminating Tetranichus Urticae. And result of experimental field showed over 85%, 71%, 70% succes rate within 7day.And biotic pesticide was shower stringer activity against hatching rate, trapnest of Tetranichus Urticae.

Interdisciplinary Program of Graduate School for Bioenergy & Biomaterials, Chonnam National University, Gwangju, 500-757, Philos & D Inc., Gwangju, 500-706. 1Interdisciplinary Program of Graduate School for Bioenergy & Biomaterials, Chonnam National University, Gwangju, 500-757. 2Interdisciplinary Program of Graduate School for Bioenergy & Biomaterials, Chonnam National University, Gwangju, 500-757, Korea Institute of Petroleum Management, Gwangju, 506-020. 3School of Biological Sciences and Technology, Interdisciplinary Program of Graduate School for Bioenergy & Biomaterials, Function Food Research Center, Biotechnology Research Institute, Research Institute for Catalysis, Engineering Research Institute, Chonnam National University, Gwangju, 500-757. Biodiesels are alkyl esters produced by transesterification process of triacylglycerols with methanol and catalysts. The cold filter plugging point of biodiesels depend on the raw material used. The high CFPP value of biodiesel is a constraint to use in diesel engine at low temperature. In this study an attempt has been made to reduce the CFPP value by blending jatropha-biodiesel and rapeseed-biodiesel. We got the following empirical equation for blending by the result. Y = aX ＋ b

Clostridium beijerinckii is a gram-positive, spore-forming, obligate anaerobic bacterium, which is able to produce solvents acetone, butanol and ethanol by using various sugars. A gene expression reporter system (pBEst-MTL) for C. beijerinckii NCIMB 8052 was developed by using esterase gene from Bacillus subtilis as a reporter gene. In order to test the reporter system, promoters of two key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), were cloned upstream of the reporter gene in pBEst-MTL in order to construct plasmids pBEst-MBP and pBEst-MBT, respectively. Detection of esterase activity in time course studies performed with strains NCIMB 8052 [pBEst-MBP], NCIMB 8052 [pBEst-MBP] demonstrated that the reporter gene produced a functional esterase in C. beijerinckii. In addition, time course studies revealed differences in esterase specific activity profiles of strains NCIMB 8052 [pBEst-MBP], NCIMB 8052 [pBEst-MBP], suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters.

Formate is generated from pyruvate and converted to carbon dioxide and hydrogen by formate hydrogen lyase (FHL). FHL enzyme consists of many proteins including two formate dehydrogenase H (FDH-H) and hydrogenase (Hyd3). This enzyme complex is responsible for hydrogen production in E. coli cells during fermentation of organic carbons. In order to improve the activity of FHL in whole cell, a series of E. coli mutant strains were constructed using E. coli BW25113 as parent strain. First of all, hyaAB and hybBC, encoding hydrogenase 1 and hydrogenase 2, respectively, which catalyze hydrogen uptake reactions, were disrupted. Then, ldhA, encoding lactate dehydrogenase and frdAB encoding fumarate reductase were deleted to construct the resulting strain, SH5 (hyaAB, hybBC, ldhA and frdAB). This SH5 strain was further manipulated to construct SH5-5 strain which has two more modifications from SH5: (i) Deletion of IscR, an iron-sulfur cluster repressor which reduces the transcription of iron-sulfur cluster operon, and (ii) overexpression of fhlA, a FHL transcriptional activator. The SH5-5 (ΔiscR, and up-regulated fhlA) showed an increased specific activity of 2.6 μmol H2 mg-1 min-1. In omparison, the parental E. coli BW25113 showed the specific activity of 1.2 μmol H2 mg-1 min-1 and SH5 showed1.4 μmol H2 mg-1 min-1, respectively.

A recycling culture system was introduced to maintain constant temperature for outdoor cultivation of Chlorella minutissima. A 14 ℓ photo-bioreactor was designed to recycle 35℃ warm media for outdoor long-term cultivation. For autotrophic cultivation, the media temperature was measured as 35℃, maintaining 1.60 (g-dry wt./L) of maximum biomass. This system was proved to maintain relatively constant temperature of 34℃ under fed-batch condition. However, for batch cultivation, temperature was decreased down to 21℃. It seems that batch culture would not be feasible for outdoor long-term cultivation. The mixotrophic process maintained 11.5 (g-dry wt./L) of maximum cell growth with 27 (%, w/w) of total lipid under recycling culture system. Therefore, based on this result, we can expect this recycling culture system to be applied for long-term outdoor cultivation even in Korea where there is relatively low temperature season for several months.

Lignocellulosic materials are regarded as an alternative energy source for bioethanol production to reduce our reliance on fossil fuels. Pretreatment is important for improving the enzymatic digestibility and fermentation yield. In this study, barley straw was pretreated by using NaOH, to produce bioethanol. And by using RSM(Response urface Methodology), the optimum conditions of pretreatment was investigated. The independent variables for pretreatment were selected as pretreatment temperature, pretreatment time and NaOH concentration. The determined optimum condition is pretreatment temperature ; 129.02°C, pretreatment time ; 3.23h, NaOH concentration ; 12.58% (w/w, NaOH/Biomass). The results are as follow : Delignification ; 71.50%, Glucose concentration ; 31.61g/L, Ethanol concentration : 15.36g/L. This results may provide useful information with regard to the development of more economic and efficient bioethanol production.

1,3-propanediol (1,3-PD) is of commercial attention as an important monomer to synthesize a new type of polyester, polytrimethylene terephthalate (PTT) which has excellent properties in textile and fiber industries. Kebsiella pneumoniae is an excellent 1,3-PD producer, but the simultaneous production of many by-products (2,3-butandiol, Ethanol, Succinic acid, etc.) have greatly reduced the fermentation efficiency of 1,3-PD. Hence, to eliminate the by-products synthesis, the oxidative branch of glycerol metabolism was inactivated by deleting the genes involved in the synthesis of by-products from the chromosomal DNA. The present investigation focuses on the effect of the culture conditions on the production of 1,3-PD from glycerol using the engineered strain. The fermentation parameters, such as pH control, aeration volume and cell concentration, were examined in fermentor-scale experiments.

The pathogenicity of the insect-parasitic nematode Butlerius sp. ANU501 was evaluated against three major cruciferous insect pests: Artogeia rapae (Lepidoptera: Pieridae), Mamestra brassicae (Lepidoptera: Noctuidae), and Plutella xylostella (Lepidoptera: Yponomeutidae). All insect species were highly susceptible to Butlerius sp. ANU501 and the larval mortality increased with an increasing dosage of nematodes under petri dish experiment. The pathogenic effect of nematodes was higher for the 4th instars than those for the corresponding 3rd instars in the order of P. xylostella, A. rapae, and M. brassicae, with the highest mortalities of 93.5, 88.2, and 77.8%, respectively. LD50 values of Butlerius sp. ANU501 against 4th instars of P. xylostella, A. rapae, and M. brassicae were 33.1, 36.1, and 48.2, and LT50 of 33.6, 41.1, and 61.5 h, respectively. The nematodes established in host varied significantly with insect species in P. xylostella, A. rapae, and M. brassicae (e.g., 6.9, 4.1, and 3.7 at a dose of 40 nematodes per larva for 3rd instars and 8.9, 5.7, and 4.6 at 80 nematodes per larva for 4th instars). Overall, Butlerius sp. ANU501 caused higher pathogenicity in P. xylostella, than in the other insect species.

Nanoparticles such as carbon, iron, silicon, silver and titania (TiO2) are now closely related with current evironment and human life. Several studies indicated that nanomaterials are likely to harmful to biological system. To evaluate and investigate the toxicity of silver and titania, Saccharomyces cerevesiae was used for model organism. The effect of silver on the lethality of S. cerevisiae showed both concentration and size dependent manner. Whereas titania rely on the concentration and light exposure by FACS analysis. Global pattern by proteomic study result in wide range of protein expression. These results suggested that silver and titania affect not only oxidative or metal stress but also metabolism.

1,3-propanediol (1,3-PD) is of commercial attention as an important monomer to synthesize a new type of polyester, polytrimethylene terephthalate (PTT) which has excellent properties in textile and fiber industries. Klebsiella pneumoniae is an excellent 1,3-PD producer, but the simultaneous production of many by-products (2,3-butandiol, Ethanol, Succinic acid, etc.) have greatly reduced the fermentation efficiency of 1,3-PD. Recently we engineered mutant strains of K. pneumoniae to eliminate the formation of by-products hindering the downstream process, typically 2,3-butanediol, during the production of 1,3-propanediol (1,3-PD) from glycerol by inactivating the oxidative branch of the glycerol metabolic pathway. Although the by-product formation was successfully eliminated, production yield and productivity of 1,3-PD was severely diminished in the mutant strains. This study presents optimization of medium component to improve the 1,3-PD production using the recombinant strain defective of the glycerol oxidative pathway eliminating by-product formation

The pathogenicity of Butlerius sp. ANU501 against three major cruciferous insect pests: Artogeia rapae (Lepidoptera: Pieridae), Mamestra brassicae (Lepidoptera: Noctuidae), and Plutella xylostella (Lepidoptera: Yponomeutidae) was evaluated. In pot and greenhouse experiment, the larval mortality increased with an increasing dosage of nematodes. The efficacy in greenhouse varied with vegetable species, depending on the persistence time of nematode suspension on the vegetable leaves after spraying. Just after spraying nematodes, the remaining water content and live nematode number on leaf were 7.09 g and 49.6 nematodes for the vegetable Brassica lee ssp. Namai., and 6.37 g and 50.2 nematodes for Brassica junacea L., whereas values for Brassica oleracea L. var. acephala Alef. (D.C.) were very low, 4.16 g and 6.3 nematodes. In Brassica lee ssp. namai., Butlerius sp. ANU501 caused the highest efficacy against P. xylostella (88%), A. rapae (29%), and M. brassicae (37%), when applied at a rate of 3 x 1010 nematodes/ha. Butlerius sp. ANU501 did not provide satisfactory control for A. rapae and M. brassicae, but were significantly effective in control of P. xylostella. This study shows that Butlerius sp. have the great potential as a biological control agent for P. xylostella in greenhouse.

The pathogenic effect of the insect-parasitic nematode Butlerius sp. ANU501 on insect pests and the nematode reproductive capacity in insect larvae were evaluated under laboratory conditions. Ten days after nematode treatment, larval mortalities of 70-94 % were obtained for fourteen species of insect pests of the order Coleoptera and Lepidoptera. The highest mortality of 94.4% was observed for Blitopertha orientalis larvae from which the nematode had been originally isolated. The nematode reproductive yield in insect cadavers varied with insect species from 2.6 x 103 - 35.8 x 103 nematodes per host. High nematode yields of over 30 x 103 were obtained for larvae of B. orientalis, Dichocrocis punctiferalis, Galleria mellonella, and Palpita indica. Butlerius sp. ANU501 is a good candidate as a pest control agent for houses and/or golf courses. For application, field tests should be performed.

The proinflammatory cytokines, IL-1β and TNF-α, are known to play a key role in inflammatory reaction in many forms of acute and chronic neurological diseases. In Parkinson’s disease (PD), those cytokines immunoreactivity was significantly increased in the substantia nigra and striatum. Moreover, it is implicated that the production of those cytokines from activated microglial cells in Parkinson’s brain contributes to the increase of pathological oxidative stress. However, it is not clear what is the molecular mechanism of cytokines-related dopaminergic cell death underlying PD. Of interest, treatment with IL- 1β and TNF-α induced minimal cell death when compared with other PD-related neurotoxicants. Although IL-1β induced a transient increase in reactive oxygen species (ROS) production within an hour, TNF-α did not increase ROS production even within 12 hours in dopaminergic neurons. The transcription factor, NF-κB was translocated into nucleus in response to IL-1β prior to ROS increase in dopaminergic cells. In addition, IL-1β activated the JNK 1/2 moderately in dopaminergic neurons and triggered the dopaminergic cell death.

An experiment was carried out to compare wound healing processes from the porcine skinderived and human-derived wound dressings. A common treatment of wounds is wound dressing derived from human cadaver skins which were distributed by approved tissue banks. The wound dressings are processed for hospital uses under the strict guidelines. The supply of human skins is limited. Therefore, we examined possibility of an alternative source of processed wound dressing derived from porcine skin. Porcine skins were procured using Dermatome (Zimmer Inc.) and manufacturing process was followed as human cadaver skin (Hans Biomed Procedures). Final dressing and intermittent processing materials were examined to compare with human skin in physical and biochemical properties. And a beagle dog was used in vivo experiment, induced wounds and applied dressing materials from both porcine and human. Structural and physical properties of the two skin dressings were showed similar by SEM, histological analysis, IHC, tensile strength, collagenase decomposition and GAGs assay. Also in an animal experiment, wound healings were taking place about the same degree of contraction and epithelialization whether porcine or human originated materials applied. In conclusion, porcine skin-derived dressing may be found to have equivalent efficacy and effects as the human skin-derived. We suggest that wound dressing derived from porcine skin can be used, when human skinderived dressings are not available and manufactured under the proper guideline.

Endoplasmic reticulum (ER) stress initiates apoptotic processes, mediated by activated caspase-12 in rodent. Caspase-12 is located in ER as a latent zymogen and is activated by pharmacological ER stress inducers such as thapsigargin (inhibitor of ER Ca++-ATPase) and tunicamycin (inhibitor of glycosylation). However, it is not known if caspase-3 is a major downstream caspase of ER stress in dopaminergic neurons. In this study, we used caspase-3(-/-) knockout mutant dopaminergic cells to prove the direct relationship between caspase-3 and caspase-12. Of interest, physiological blockade of caspase-3 activity did not induce the accumulation of caspase-12 in dopaminergic neurons. In addition, compensatory activation of caspase-9 and other caspases had not occurred in caspase-3(-/-) mutant cells during ER stress-induced cell death. Our results suggest that caspase-3 may not be a major downstream executioner caspase in the cascade of caspase-12-related ER stress process in dopaminergic neurons.

Silk fibroin from Bombyx mori was sulfated in an aqueous sulfuric acid as a solvent and a catalyst. Incorporation of sulfate into the fibroin molecules was verified by FT-IR and a simultaneous hydrolysis of fibroin was observed by measuring its molecular weight distribution using a gel permeation chromatography. Unlike to fibroin used as a substratum for cultivation of animal cells [1], the sulfated fibroin decreased the growth rate of 3T3 cells in DMEM/Ham' s F12(1:1) media containing 10% FBS. Heparin, a sulfated peptide, inhibited the attachment and growth of Balb/c-3T3 fibroblasts [2]. Thus, the effect of fibroin, enzymatically hydrolyzed fibroin, acid-hydrolyzed fibroin and sulfated fibroin were investigated on the attachment, metabolic activity and growth of 3T3 cells in this report. Sulfated fibroin at the concentration of 0.1 % (w/v) in the culture media reduced the specific growth rate by 20% but other fibroin peptides showed varying responses to the levels of peptides supplied. Fibroin has been used as a 3D scaffolds for cell cultivation [3]. A composite of fibroin and sulfated fibroin is under investigation as a potential extracellular matrix interacting cell adhesion and growth control.

Alopecia is not life threatening, but patients who undergo alopecia often experience severe mental stress. In addition, the number of individuals afflicted by alopecia has been increasing steadily. The most effective treatment of alopecia developed to date is auto hair transplantation. To overcome the limitations associated with current therapies for the treatment of alopecia, many researchers have attempted to revive hair follicles by in vitro culture of hair follicle cells and subsequent implantation in the treatment area. Previously, we demonstrated that umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be isolated and expanded successfully from the Wharton's Jelly. Culture-expanded UC-MSCs formed aggregate similar to native dermal papilla (DP) in special media (DPFM), and reconstructed dermal papilla like tissues (DPLTs) could induce new hair follicles. The purpose of the present study was to optimize the reconstruction of DPLTs. As in the case of MSCs, when compared to differentiated cells, DPLTs require an additional step to induce differentiation into dermal papilla cells. However, it is necessary to use hepatocyte growth factor (HGF) in the differentiation step, which is relatively expensive. To reduce the expenses associated with cell therapy using MSCs, it is necessary to optimize this differentiation step. To accomplish this, we evaluated the effects of various cells inoculation densities and growth factors during differentiation.

While there is clear evidence that granule and periglomerular cells of olfactory bulb originate in the anterior subventricular zone (SVZa), it is not known when they acquire the potential to become dopaminergic neurons. In this study, we investigated this issue utilizing a transgenic mouse with high level, tissue specific expression of lacZ under control of 9kb tyrosine hydroxylase (TH) promoter. Transgene expression was found in the neonatal, but not in the adult, rostral migratory stream (RMS) suggesting differential dopaminergic phenotypic regulation in developing and mature animals. Migrating cells were labeled using the lacZ gene expression kit and then substrate was delivered with the influx reagent and observed by 2-photon microscopy. Labeled cells were found in the RMS, glomerular and mitral cell layers between postnatal day 2 (P2) and P7. The adult RMS did not contain either THor β-gal-immunostained progenitor cells. These results suggest that dopaminergic phenotypic determination may finish in the developing neonatal stage.

In this study we are willing to recognize the effect of the 2D and 3D mechanical tension on the proliferation and osteogenesis of human dental pulp stem cells (DPSCs). In the 2D tension system, we evaluated the proliferation and extracellular matrix (ECM) production of DPSCs using a flexwell system that imposed cyclic mechanical tension at 0.03 Hz with 0, 5, and 8 % strains. In the early stage (4 days), DPSCs at 5 and 8 % strains had a similar proliferation, which was higher than the control. However in the late stage (10 days), DPSCs at 8 % strain had a higher proliferation than the control and 5 % strains. RT-PCR analysis was shown that mechanical tension increased collagen and osteopontin expression. In the 3D tension system, we evaluated the osteogenesis of DPSCs on the silk scaffold using a bioreactor with 10% strain. The tension was applied with 0.2 Hz during 5 days and continuously applied during 10 days. Application of 10% tension culture reported in increases in collagen type I, fibronectin, osteoprotegerin, bone sialoprotein. These data suggest that mechanical stimulation can serve as a potent positive modulator of proliferation and promote osteogenesis of DPSCs.

Current methods for quantitatively comparing complex proteomes such as two-dimensional gel electrophoresis (2-DE), fluorescent 2-D difference in gel electrophoresis (2-D DIGE), and liquid chromatography (LC)-mass spectrometry (MS) have limited resolution and dynamic ranges, and consequently can detect only a small portion of complex proteomes. Therefore, sample prefractionation is critical for more comprehensive coverage of the proteome and reliable detection of low abundant proteins. In this study, we report a 3-D DIGE method using DIGE labeling combined with microscale solution isoelectric focusing (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. To illustrate its utility, this 3-D DIGE method was applied to analysis of human melanoma cell lines and mouse lung tissue extracts. This method has the advantage of much more extensive proteome coverage with detection of proteins over a wider concentration range compared with conventional 2-D DIGE alone. In addition, the use of fluorescent labeling prior to MicroSol- IEF avoids any complications resulting from slight run-to-run variations during MicroSol-IEF fractionations or the subsequent 2-D gel separations. Thus the combination of MicroSol-IEF and 2-D DIGE into a 3-D DIGE strategy produces a powerful method for more comprehensive and quantitative comparison of protein profiles of very complex proteomes.[This work was supported by National Institutes of Health Grants CA93372 and CA92725, as well as institutional grants to the Wistar Institute including an NCI Cancer Core Grant (CA10815), and grants from the Pennsylvania Department of Health]

Melanoma is an excellent model to study molecular mechanisms of tumor progression because melanoma usually develops through a series of architectural and phenotypically distinct stages that are progressively more aggressive, culminating in highly metastatic cells. In this study, in order to identify the candidate proteins involved in tumor progression and to better understand key pathways underlying melanoma metastasis, comparative proteome analysis on two melanoma cell strains with low and high metastatic potentials, WM793 and 1205LU, respectively was performed using a 3-D DIGE method consisting of sample Cydyeslabeling, microscale solution isoelectric focusing (MicroSol-IEF) prefractionation, and 2-dimensional differential gel electrophoresis (2-D DIGE). This method identified 157 protein spots compared with only 15 spots using 2-D DIGE, which showed abundance changes that were both statistically significant and at least 2-fold. Analysis of these gel spots using LCMS/MS resulted in unambiguous identification of the major protein responsible for the observed abundance change in 156 cases. These 156 identifications represented 110 unique proteins because some spots were different isoforms of the same protein. As expected, some proteins observed in this study were previously linked to metastasis in either melanoma or other tumor types, but in addition, many novel proteins were identified that had not previously been implicated in melaoma metastasis. Most of the novel observed abundance changes closely correlate with biological processes central to cancer progression, such as cell death and growth, and tumorigenesis. In addition, the vast majority of protein changes mapped to six cellular networks, which included known oncogenes (JNK, c-myc, and N-myc) and tumor suppressor genes (p53 and TGF-β) as critical components. These six networks showed substantial connectivity, and most of the major biological functions associated with these pathways are associated with tumor progression. Thus this study led to novel insights into cellular pathway implicated in melanoma metastasis. [This work was supported by National Institutes of Health Grants CA93372 and CA92725, as well as institutional grants to the Wistar Institute including an NCI Cancer Core Grant (CA10815), and grants from the Pennsylvania Department of Health]

Factor X, a vitamin K-dependent blood coagulation glycoprotein, is the precursor for factor Xa, the enzyme component of the prothrombinase complex. The factor X is cleaved during coagulation by several different protease including the intrinsic Xase complex, the factor Xactivating enzyme from Russell's viper venom(RVV) and trypsin, and also by extrinsic pathway to give an active enzyme factor Xa. Factor Xa is often used to remove fusion tags from expressed proteins. Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile- (Glu or Asp)-Gly-Arg. Human factor X was produced in transgenic rice cell suspension culture with the rice amylase 3D promoter. RAmy3D promoter is highly expressed by sugar starvation. The expression of human factor X as mRNA and protein in transgenic rice cell was detected Northern and Western blot analysis. The enzyme activity of RVV activated human factor X was generally characterized by using EGFP-hTNFa fusion protein as substrate, which contained a human factor Xa cleavage site between two moieties. These results demonstrate the use of human factor Xa derived from plant cell suspension culture as proteases (This work was supported by the grant from Korean Ministry of Knowledge Economy)..