Abnormal prions are infectious agents involved in a neuro-degenerative disease, which occurs naturally such as Chronic Wasting Disease (CWD) in deer and elk, Bovine Spongiform Encephalopathy (BSE) in cattle, Scrapie in sheep and goats and Creutzfeldt-Jakob Disease (CJD) in humans. The cellular prion protein of the elk consists of 233 amino acids (residues 25-257), which represents an autonomous folding unit with three α-helices and two-stranded anti-parallel β-sheets. Here, we demonstrated elk-recPrP (Elk recombinant prion protein) which can be obtained as follows; (1) Cloning of elk PrP gene, (2) Expression of a histidine-tagged full-length elk PrP by induction with IPTG in E. coli and (3) Purification by affinity chromatography using Ni-NTA agarose resin. In Western blot and ELISA analysis, elk-recPrP showed specific activity against anti-PrP monoclonal antibody. Thus, our elk-recPrP would be a useful tool for the understanding of basic structure and mechanism studies of PrPSC formation.

The use of bacteria in the treatment of cancer has a long and interesting history. The use of live bacteria in this way however has a number of potential problems including toxicity. Purified low molecular weight bacterial proteins have therefore been tested as anticancer agents to avoid such complications. Oral cancer is a widely occurring disease around the world and these lesions are typically very resistant to anticancer agents. In our present study we investigated the effects of purified recombinant azurin from Pseudomonas (P.) aeruginosa against YD-9 (p53-positive) human oral squamous carcinoma cells. Azurin showed cytotoxic effects against these cells in a dose dependent manner. The cell death accompanied by this treatment was found to be characterized by chromatin condensation and apoptotic bodies. Azurin treatment was further found to increase the expression of p53 The stabilization of p53 and induction of apoptosis in YD-9 cells by azurin suggests that it has potentially very strong anticancer properties in oral squamous carcinoma.

A new Bacillus thuringiensis isolate 19-22 (Bt 19-22) exhibited high anti-fungal activity against barley powdery mildew (Blumeria graminis f. sp. hordei). The cry gene content of Bt 19-22 comprised cry1Aa, cry1Ab, cry1Ac and cry1D which have high insecticidal activity against lepidopteran larvae. We tried to confer a dipteran insecticidal activity to Bt 19-22 for constructing a recombinant strain which has multiple functions, anti-fungal and dual insecticidal activity. The insecticidal cry11Aa gene of B. thuringiensis was constructed under cry1Ac promoter in an E. coli-B. thuringiensis shuttle vector (pPro11A). The plasmid, pPro11A was introduced into Bt 19-22 isolate by electroporation and four transformants which had different cry gene contents were identified by PCR with cry11Aa and cry1-type specific primers. Among them, a Bt 19-22 transformant (11A/19-22 No. 7) expressed Cry11A protein (approximately 70 kDa) successfully without change of its inherent characteristics such as Cry protein expression and antifungal activity. The insecticidal activity of 11A/19-22 No. 7 was checked against Plutella xylostella and Culex pipiens. These results suggests that the recombinant strain shows dual insecticidal activity against lepidopteran and dipteran larvae as well as antifungal activity.

This study was conducted to investigate the insecticidal capacity of several recombinant baculoviruses to P. xylostella and S. exigua larvae. NeuroBactrus was constructed as follows: the cry1-5 of Bacillus thuringiensis 2385-1 was inserted into Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of polyhedrin gene promoter, and insect-specific neurotoxin from the scorpion Androctonus australis (AaIT) under the control of early promoter from Cotesia plutellae bracovirus was introduced by fusion of orf603 partial fragment in the opposite direction of polyhedrin gene, respectively. Other recombinant baculoviruses derived from the NeuroBactrus - NBt-DelA (deleted AaIT), NBt-Del5 (deleted cry1-5), and NBt-DelA5 (deleted AaIT and cry 1-5) - were manufactured in serial passages in vitro. The data were analyzed by SPSS. The value of LC50 was lower when P. xylostella larvae fed on cabbage coated with NeuroBactrus (4068.4) than when it fed on cabbage coated with AcMNPV (4.5x106). Survival time (ST50) of P. xylostella larvae (2.54days) was shorter when it fed on cabbage coated with NeuroBactrus than when it fed on cabbage coated with other recombinant baculoviruses (7.54days, 7.68days, and 8.26days) and AcMNPV (9.67days). S. exigua larvae presented the same results.

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked α- and β-subunits. To study the function and signal transduction of tethered recombinant-eCG (rec-eCG), a single chain eCG molecule was constructed, and the rec-eCG protein was prepared. In this study, we constructed 5 mutants (Δ1, Δ2, Δ3, Δ4, and Δ5) of rec-eCG using data about known glycoprotein hormones to analyze the role of specific follicle stimulating homone (FSH)-like activity. Three amino acids of certain specific sites were replaced with alanine. The expression vectors were transfected into CHO cells and subjected to G418 selection for 2～3 weeks. The media were collected and the quantity of secreted tethered rec-eCGs was quantified by ELISA. The LH- and FSH-like activities were assayed in terms of cAMP production by rat LH/CG and rat FSH receptors. Then, the metabolic clearance rate analyzed by the injection of rec-eCG (5 IU) into the tail vein was analyzed. The mutant eCGs (Δ1, Δ4, and Δ5) were transcripted, but not translated into proteins. Rec-eCG Δ2 was secreted in much lower amounts than the wild type. Only the rec-eCG Δ3 (β-subunit: Gln94-Ile95-Lys96→Ala94-Ala95-Ala96) was efficiently secreted. Although activity is low, its LH-like activity was similar to that of tethered eCGβα. However, the FSH-like activity of rec-eCGβαΔ3 was completely flat. The result of the analysis of the metabolic clearance rate shoed the persistence of the mutant in the blood until 4 hours after the injection. After then, it almost disappeared at 8 hours. Taken together, these data suggest that 94～96 amino acid sequences in eCG β-subunit appear to be of utmost importance for signal transduction of the FSH receptor.

Recently, the genome of Spodoptera litura granulovirus (SlGV) which encodes 133 putative open reading frames (ORFs) was completely sequenced. In this study, to screen novel insecticidal genes of SlGV, we first constructed an advanced plasmid capture system, pPCS-TPI, which contains not only pUC19 ori and ampicillin resistance gene but also Autographa californica nucleopolyhedrovirus (AcMNPV) ORF603 and ORF1629 homologous region between Tn7L and Tn7R. In order to introduce genomic segments of SlGV into the genome of AcMNPV, genomic DNA of SlGV was digested with EcoRI and self-ligated. These self-ligated segments were in vitro transposed with the pPCS-TPI donor by the help of TnsABC* transposase. By this, 10 EcoRI-digested genomic segments of the SlGV were cloned, and these clones were co-transfected with the bApGOZA DNA into sf9 cells to generate corresponding recombinant virus, respectively. The resulting recombinant viruses harboring genomic segments of the SlGV could be used to investigate the insecticidal activity and/or other functions originated from the introduced genomic segments of the SlGV.

Sorghum-Sudan grass which is an annual forage grass in Gramineae have been bred for biomass promotion. The recombinant inbred line (RIL) populations were derived from the F2:3 genetic mapping population of Sorghum 314A×zongke Sudan grass. The breeding selection based on agronomic characters and DNA molecular marker statistical analysis was carried among the progenies of the RIL populations, and 9 recombinant breeding progenies were determined as heterobeltiosis with high yield. According to DNA diversity analysis of simple sequence of repeats(SSR) marker, the genetic varieties were abundant among the families, and the recombinant breeds of high yield were distributed among the different RIL populations.2 family lines, which have similar agronomic characters and yield, and whose genetic distance nearly equal to zero, showed difference on arid tolerance. 4 breeding lines were selected on agronomic characters and resistance in the field trials, and 2 breed lines were qualified for the regional testing.

Human recombinant IL-32 induces the production of large amounts of several proinflammatory cytokines and chemokines, even in macrophage cell lines by activation of NF-κB and MAPK. The ability of IL-32 to potentiate inflammation is not the sole action of this cytokine. IL- 32 stimulates prostaglandin (PG)E2 in human PBMCs, which are pivotal in inflammation. The development of recombinant protein consisting of IL-32 is of great medical and industrial importance. Agrobacterium strains GV3101 harboring pCAMBIA1304 include IL- 32 vectors were used to infect the mushroom fruiting body pieces. For transformation experiments, fruiting body gill tissue pieces were vacuum infiltrated with the suspension of induced bacteria until the air had been completely purged. Mushroom mycelium appeared at the margins of the tissue pieces after 9 to 14 days on selection medium with hygromycin at 50㎍/㎖. Transformants PCR analyses confirmed that the IL-32 gene was into the genome of Pleurotus eryngii. The present results demonstrating the usefulness of the transgenic technique s in the genetic manipulation and improvement of mushroom.

The aim of this study is to evaluate the estrogenic activity of Cucurbita pepo seed extract which includes β-sitosterol and other phytosterols. Sample was extracted from Cucurbita pepo seed by supercritical carbondioxide method and resuspended with ethanol. Estrogenic activity was measured by recombinant yeast assay which detects estrogenic activity using recombinant yeast with high level of estrogenic receptor. However, estrogenic activity of pumpkin seed extract was not found in this study. Based on this data, pumpkin seed extract will not cause estrogenic disturbance.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties such as higher insecticidal activity and recovery to wild-type baculovirus. For this, Bacillus thuringiensis crystal protein gene (cry1-5) was introduced into Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrincry1- 5-polyhedrin under the control of poyhedrin gene promoter. In the opposite direction of this fusion gene, an insect-specific neurotoxin gene (AaIT) under the control of early promoter from Cotesia plutellae bracovirus was introduced by fusion of orf603 partial fragment. Western hybridization and confocal microscopy revealed that AaIT neurotoxin and Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus and that the fusion protein occluded into the polyhedra. In addition, the fusion protein was activated as about 65 kDa of crystal protein when treated with trypsin. The NeuroBactrus showed high level of insecticidal activity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Re-recombinants derived from the NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro and in vivo. These results suggested that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 fragments.

Recently, we constructed a novel recombinant baculovirus genome, bEasyBac, enabling easy and fast generation of pure recombinant baculovirus without any purification step. In the bEasyBac, bacteriophage lambda site-specific attachment (att) sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus (CpBV) ORF3005 early promoter to negatively select against non-recombinant background. The bEasyBac could replicate in host insect cells only when the barnase gene was replaced to gene of interest by in vitro transposition. When the bEasyBac was transposed with pDualBac-EGFP and the EGFP expression efficiency along passage was investigated, the resulting recombinant virus, EasyBac-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, AcEGFP, which was constructed using bAcGOZA system, whereas, the non-purified AcEGFP showed quite reduced level of EGFP along passages. Moreover, no non-recombinant backgrounds were detected from unpurified EasyBac-EGFP stocks. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established. These results suggest that the bEasyBac has an effective benefit enabling for high-throughput baculovirus expression vector without purifying recombinant virus.

To develop an advanced baculovirus insecticide with additional advantages, such as higher toxicity and recovering to wild-type baculovirus, a novel recombinant baculovirus, NeuroBactrus was constructed. Bacillus thuringiensis crystal protein gene (cry1-5) and an insect-specific neurotoxin gene (AaIT) were introduced into Autographa californica nucleopolyhedrovirus genome by fusion of polyhedrin-cry1-5-polyhedrin under the control of poyhedrin gene promoter, and by fusion of orf603 partial genes and AaIT under the control of early promoter of ORF3006 from Cotesia plutellae bracovirus. About 150 kDa of Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by NeuroBactrus was occluded into the polyhedra, and activated as about 65 kDa of crystal protein when treated with trypsin. RT-PCR analysis indicated that transcription of AaIT gene occurs by 2 h postinfection (p.i.) and increased at 16 h p.i.. NeuroBactrus showed high toxicity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcNPV. Re-recombinants derived from NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro. This result showed that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 genes.

A new Bacillus subtilis isolate showed high anti-fungal activities (more than 80% control efficacy) against several plant diseases such as rice blast (Magnaporthe grisea), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans) and wheat leaf rust (Puccinia recondita). We tried to confer an insecticidal activity to this B. subtilis isolate for constructing a recombinant strain which has dual functions, anti-fungal and insecticidal activity. The insecticidal cry1Ac gene of B. thuringiensis was constructed under its own promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K-1Ac). The plasmid, pHT1K-1Ac was introduced into B. subtilis isolate by electroporation and the transformant was confirmed by PCR with cry1Ac specific primers. B. subtilis transformant produced a parasporal inclusion in the cells as in B. thuringiensis and the size of that protein was appox. 130 kDa. The insecticidal activity of the transformant was checked against lepidopteran pest, Plutella xylostella. This result suggests that this recombinant B. subtilis strain shows the possibility of controlling harmful insect pests as well as plant fungal diseases simultaneously at one crop, and both culture broth and harvested cells of this strain can be used as individual biological control agents separately for integrated crop protection.