Hyouk Joung

Anti-complementary activity of protostane-type triterpenes fromAlismatis rhizoma

Four protostane-type triterpenes, alisol B 23-acetate (1a), alisol C 23-acetate (2a), alisol B (3a), and alisol A 24-acetate (4a), were isolated from the rhizome ofAlismatis plantago-aquatica L. var.orientate Samuelson (Alismataceae) and eleven protostane derivatives (compounds1-11) were obtained by selective modification from alisol B 23-acetate (1a). These compounds were investigated for their anti-complement activity against the classical pathway of the complement system. Alisol B (3a) and alisol A 24-acetate (4a) exhibited anti-complement activity with IC50 values of 150 and 130 μM. Among the synthetic derivatives, the tetrahydroxylated protostane triterpene (9) showed moderate inhibitory activity with IC50 value of 97.1 μM. Introduction of an aldehyde group at C-23 (10; IC50 value, 47.7 μM) showed the most potent inhibitory effect on the complement systemin vitro

Effect of sealed and vented gaseous microenvironments on the hyperhydricity of potato shoots in vitro

Abstract Different types of ventilation of the culture vessel with and without the ethylene inhibitor KMnO 4 in the culture media were investigated in terms of their effect on hyperhydricity of in vitro cultured potato shoots. The shoots grown in a completely sealed vessel (CSV) were severely vitrified, but the shoots cultured in a gas permeable vessel (GPV) showed normal shoot proliferation. Dry weight percentage and chlorophyll content were lower in the vitrified shoots than in the normal shoots. The levels of ethylene and CO 2 were significantly higher in the CSV, but little ethylene was detected in the GPV system. When an ethylene scrubber (perlite treated with KMnO 4 ) was applied to the CSV, the shoots exhibited normal growth without any sign of vitrification. In addition, no ethylene was found in CSV with the ethylene scrubber (CSVE). These results indicate that hyperhydricity is related to the ethylene accumulated in the culture vessel during the in vitro culture of potato shoots. It is suggested to use CSVE to reduce the ethylene level and overcome hyperhydricity.

Transgenic tomatoes expressing human beta-amyloid for use as a vaccine against Alzheimer’s disease

Human β-amyloid (Aβ) is believed to be one of the main components of Alzheimer’s disease, so reduction of Aβ is considered a key therapeutic target. Using Agrobacterium-mediated nuclear transformation, we generated transgenic tomatoes for Aβ with tandem repeats. Integration of the human Aβ gene into the tomato genome and its transcription were detected by PCR and Northern blot, respectively. Expression of the Aβ protein was confirmed by western blot and ELISA, and then the transgenic tomato line expressing the highest protein level was selected for vaccination. Mice immunized orally with total soluble extracts from the transgenic tomato plants elicited an immune response after receiving a booster. The results indicate that tomato plants may provide a useful system for the production of human Aβ antigen.

Role of genetic factors and environmental conditions in recombinant protein production for molecular farming.

Plants are generally considered to represent a promising heterologous expression system for the production of valuable recombinant proteins. Minimal upstream plant production cost is a salient feature driving the development of plant expression systems used for the synthesis of recombinant proteins. For such a plant expression system to be fully effective, it is first essential to improve plant productivity by plant biomass after inserting genes of interest into a suitable plant. Plant productivity is related closely to its growth and development, both of which are affected directly by environmental factors. These environmental factors that affect the cultivation conditions mainly include temperature, light, salinity, drought, nutrition, insects and pests. In addition, genetic factors that affect gene expression at the transcriptional, translational, and post-translational levels are considered to be important factors related to gene expression in plants. Thus, these factors influence both the quality and quantity of recombinant protein produced in transgenic plants. Among the genetic factors, the post-translational process is of particular interest as it influences subcellular localization, protein glycosylation, assembly and folding of therapeutic proteins, consequently affecting both protein quantity and biological quality. In this review, we discuss the effects of cultivation condition and genetic factors on recombinant protein production in transgenic plants.

Ectopic expression of pepper CaPF1 in potato enhances multiple stresses tolerance and delays initiation of in vitro tuberization

Ethylene-responsive factors (ERFs) are plant-specific transcription factors, many of which have been linked to plant defense responses. However, little is known about the functional significance of ERF genes in potato plants compared to the model plant species Arabidopsis. We show here that overexpression of CaPF1, an ERF/AP2-type pepper transcription factor gene, effectively increased tolerance to freezing, heat, heavy metal, and oxidative stress in potatoes. Interestingly, CaPF1 was involved in tuber formation in potato plants. The time course of microtuber formation was significantly retarded in potato plants that overexpressed CaPF1 compared with wild-type potato plants. Overall, the results of the present study indicate that the pepper transcription factor gene, CaPF1, is involved in promotion of multiple stress tolerance and retardation of in vitro tuberization in potato plants.

Transgenic potato expressing Aβ reduce Aβ burden in Alzheimer’s disease mouse model

Beta amyloid (Aβ) is believed one of the major pathogens of Alzheimers disease (AD), and the reduction of Aβ is considered a primary therapeutic target. Immunization with Aβ can reduce Aβ burden and pathological features in transgenic AD model mice. Transgenic potato plants were made using genes encoding 5 tandem repeats of Aβ1–42 peptides with an ER retention signal. Amyloid precursor protein transgenic mice (Tg2576) fed with transgenic potato tubers with adjuvant showed a primary immune response and a partial reduction of Aβ burden in the brain. Thus, Aβ tandem repeats can be expressed in transgenic potato plants to form immunologically functional Aβ, and these potatoes has a potential to be used for the prevention and treatment of AD.

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins.

BackgroundPatatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.ResultsBased upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts) of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L.) was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi) vector. The CaMV 35S promoter-driven patatin hpRNAi vector was transformed into the potato cultivar Desiree by Agrobacterium-mediated transformation. Ten transgenic potato lines bearing patatin hpRNA were generated. The effects of RNA interference were characterized at both the protein and mRNA levels using 1D and 2D SDS/PAGE and quantitative real-time RT-PCR analysis. Dependent upon the patatin hpRNAi line, patatins decreased by approximately 99% at both the protein and mRNA levels. However, the phenotype (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc.) of hpRNAi lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. During glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins.ConclusionPatatin-specific hpRNAi effectively suppressed the expression of a majority of patatin variants in potato tubers via the specific degradation of individual mRNAs of the patatin multi-gene family. More importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins.

Superoxide anion regulates plant growth and tuber development of potato

A higher concentration of H2O2 was detected in the sense transgenic potato plant (SS4) with the lily chCu,ZnSOD sequence, whereas higher levels of O2− was detected in the antisense transgenic plant (SA1) than the WT plant. The elongation growth in SA1 was significantly inhibited by treatment with diphenyleneiodonium, an inhibitor of O2− generation, and promoted in the SS4 on treatment with herbicide methyl viologen, a generator of apoplastic O2−. Higher concentrations of GAs were detected during plant growth and the early stage of tuberization in SA1. Complete recovery of the above elongation growth and microtuberization pattern in transgenic plants following treatment of GA3 or an inhibitor of gibberellin synthesis, paclobutrazol, indicate that these changes were mainly caused by active GA levels. In conclusion, a specific ROS (O2− ) acts as a signal transducer via GA biosynthetic pathways for the regulation of plant growth and tuber development of potato.

High-level expression of a human β-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice

Plastid transformation has to date been applied to the expression of heterologous genes involved in agronomic traits and to the production of useful recombinant proteins. Here, we report a feasibility study for producing the human β-site APP cleaving enzyme (BACE) via transformation of tobacco chloroplasts. Stable integration of human BACE into the plastome was confirmed by PCR. Genomic Southern blot analysis detected the presence of the tobacco aadA and human BACE genes between trnI and trnA in the plastome. Northern blot analysis revealed that the aadA and BACE genes were both properly transcribed into a dicistronic transcriptional unit. Human BACE protein expression in transplastomic tobacco was determined by western blot analysis. ELISA analysis revealed that, based on a dilution series of E. coli-derived BACE as a standard, transplastomic lines accumulated BACE to levels of 2.0% of total soluble proteins. When mice were gavaged with the transplastomic tobacco extracts, they showed an immune response against the BACE antigen. The successful production of plastid-based BACE protein has the potential for developing a plant-based vaccine against Alzheimer disease.

Polyamine Biosynthesis Regulated by StARD Expression Plays an Important Role in Potato Wound Periderm Formation

An acireductone dioxygenase (ARD) gene of potatoes was isolated from the expressed sequence tags (ESTs) of potato post-suberization cDNA libraries. The highest expression levels of the StARD gene and the protein appeared 36 h after suberization. An approximate 9-fold increase in ARD activity was detected at 36 h after wounding. Real-time reverse transcription-PCR (RT-PCR) analysis and immunolocalization studies revealed that StARD transcripts increase at the wound surface of potato tubers. The polyamine (PA) contents increased significantly after wounding at the wound surface. The increased PA content and ARD activity may play an important role in wound periderm formation.