The effects of chitosan upon the experimentally induced differentiation of MDPC-23 cells, derived from mouse dental papilla cells, were investigated by RT-PCR, observations of cell morphology and Alizaline red-S staining. Chitosan was found to significantly increase and accelerate the expression of ALP mRNA but decrease the ColI transcript levels, as compared with the control, in a time-dependent manner during the differentiation of MDPC-23 cells. Chitosan also significantly downregulated ON mRNA expression and accelerated mineralization in differentiating MDPC-23 cells. These results suggest that chitosan facilitates odontoblast differentiation and mineralization and may have potential clinical applications as a dentin regeneration material.

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

Bacillus thuringiensis (B. t.) strains are important microorganism because they produced a large amount of δ-endotoxin protein per bacterial cell and their toxins are highly toxic to Lepidoptera, Coleoptera, and Diptera depending on B. t. To date, more than a hundred Cry proteins have been identified and classified into 195 holotypes, based on the amino acid sequence identity. The Cry proteins or cry genes from the Korean native B. t. isolates in this study were not identified yet. The electrospray ionization of quadrupole time of flight mass spectrometry (ESI Q-TOF MS) was used to get the internal amino acid sequences of the endotoxin-spore culture mixtures of B. t. isolates, for which polymerase chain reaction (PCR) techniques were unable to detect the cognate genes. Most of Cry proteins seperated, excized, and extracted from the one dimensional - polyacrylamide gel electrophoresis (1D-PAGE), instead of 2D-PAGE, were matched with protein databases using MS-MASCOT search program. The internal amino acid sequences which were submitted to protein BLAST (basic local alignment search tool) had partially homology with the Cry protein databases. Hence, present data strongly suggest that the de novo amino acid sequencing and ESI Q-TOF/MS analysis along with MASCOT search could be used as a simple and rapid method for detection of novel Cry toxins from B. t. isolates and identification of B. t. isolates.

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

Bacillus thuringiensis 1-3 (Bt 1-3) which was isolated from a Korean soil sample showed high insecticidal activity against Aedes aegypti as well as Plutella xylostella. The isolate was determined to belong to ssp. aizawai (H7) type by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins with a molecular weight of 130 kDa. PCR analysis with cry gene specific primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2A gene, differing from spp. aizawai (reference strain) which contains cry1Aa, cry1Ab, cry1C and cry1D. We modified the plasmid capture system (PCS) to clone plasmid from Bt 1-3 through in vitro transposition. Fifty-three clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified according to similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its 20 putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family.

Baculovirus chitinase gene (ChiA) is a late gene and is essential for liquefying host insect at the late stage of infection for its hydrolyzing chitin function. In previous report, baculovirus ChiA can offer many interseting new opportunities for pest control. Recently, a putative chitinase gene (ChiA) was identified in the Spodopter litura nucleopolyhedorvirus (SlMNPV-K1) genome. The open reading frame (ORF) contains 1,692 nucelotides (nt) and encodes a protein of 563 amino acids (aa) with a predicted molecular weight of 62.62 kDa. To conform the insecticidal activity of ChiA from SlMNPV-K1, we constructed a baculovirus transfer vector, pBac-SlChiA, and this transfer vector was co-transfected with the bApGOZA DNA into sf9 cell to generate corresponding recombinant viru which designed Ap-SlChiA. Western blot analysis indicate that SlMNPV-K1 ChiA was successfully expressed. We found the chitinase activity of recombinant virus was enhanced 53% than wide type AcMNPV by chitinase assay, and the recombinant virus showed higher evidently insecticidal activity against 3rd instar larvae of Spodotera exigua than wide type AcMNPV (4.5 time). These results suggested that the chitinase gene from SlMNPV-K1 could be successfully applied to improve pathogenicity of bauclovirus

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

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.

tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for Bursaphelenchus xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2,304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions or bands via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two non-identical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Antioxidants partially ameliorated the detrimental effects of reactive oxygen species (ROS) on sperm characteristics during in vitro storage. The objective of the present study was to investigate the single or synergetic antioxidative effect of curcumin and Vit. E on the characteristics of fresh boar sperm during in vitro storage. The sperm viability in curcumin, Vit. E supplementation and curcumin+Vit. E+H2O2 groups remained over 85.0% in 3 hr incubation period, but in 6 hr incubation period, curcumin+Vit. E+H2O2 groups was sharply dropped than those of curcumin and Vit. E group. The membrane intergrity in all evaluated groups except for H2O2 group did not significantly difference in 3 hr incubation period. The viability in curcumin or Vit. E supplementation were significantly increased than in curcumin+H2O2 and Vit. E+H2O2 group in 6 hr incubation period. The percentage of mitochondrial activity and acrosome intergrity obtained similar trends within same incubation periods irrespective of treatment. The lipid peroxidation of spermatozoal plasma membrane ranged from 11.6∼17.5 nM/l×106 and 14.0∼ 19.0 nM/l×106 in 3 hr and 6 hr incubation periods. In conclusion, curcumin or Vit. E rpplementation alone or cooperatively improved sperm viability index (motility, membrane intergrity, viability and survival rates) and fertility index (mitochondria activity, acrosome intergrity and lipid peroxidation) of fresh boar sperm, indicating that curcumin and Vit. E have a antioxidative properties through its scavenging activity against hydrogen peroxide.

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen‐thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2‐cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time‐dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

COPRISIN is an antibiotic substance extracted from Copris tripartitus. This study is intended to identify various cell biological stimuli that COPRISIN, widely known as an antibacterial substance, has on human cells and to identify its molecule mechanism. A variety of human cell lines were divided into epithelial cells including kidney cells or womb cells, and immunocyte including T cells or macrophages and, after their being cultivated and maintained, cell biological changes of the respective cells according to COPRISIN treatment were compared. As a result, it was confirmed that, different from other experiment cells, COPRISIN specifically caused cell kill in T cells and macrophages. That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to COPRISIN treatment. An Apoptosis process is one dependent upon activity of caspase family protein, it was proved that COPRISIN medium cell kill process was one through a caspase-independent route such as AIF. Though it was found out that transcription of TNF-α and extracellular TNF-α secretion increased in blood cells stimulated by COPRISIN, it was also confirmed that TNF-α was a major medium factor in a COPRISIN induced cell kill process from the fact that a cell kill process by COPRISIN was not inhibited at all with TNF-α inhibiting antibody treatment. Above results revealed that COPRISIN, different from other tissue origin cells including kidney cells, can specifically induce apoptosis in immunocyte, which is caused by a caspase-independent cell signal transmission route.

Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Here, we report for the first time the occurrence of DWV-infected bumble bees (Bombus terrestris). For the present study, the detection of DWV virus from the adult bumble bee, death adult bumble bee, mail bumble bee, pupa and larva to the infection cycle was investigated in the same colony. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from diseased insects, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

We report for the first time the occurrence of DWV-infected bumble bees (Bombus ignitus). For the present study, the detection of DWV virus from the female and male bumble bee was investigated in the same colony. The Deformed wing virus (DWV) of honeybee (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from Bombus ignitus, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV from insects, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.