Toll-like receptor 4 (TLR4) is known to contribute to the modulation of insulin resistance and systemic inflammation seen in obesity and the metabolic syndrome. The present study was performed to investigate the fertility competence of TLR4 knock out male mice (TLR4 mice) on a high-fat diet (HFD), compared to a normal-chow diet (NCD). The controls included wildtype (WT) mice fed on a HFD or NCD. Six-week-old male mice were fed with either a NCD or HFD for 20 weeks. Body and organ weights, serum levels of glucose, triglycerides and hepatoxicity, sperm quality and spermatogenesis were observed after the sacrifice. Also, randomly selected male mice were mated with virgin female mice after feeding of 19 weeks. The weight of the body and organs increased in WT and TLR4 mice on a HFD compared to those of mice on a NCD. The weights of the reproductive organs did not vary among the treatment groups. The motility and concentration of the epididymal spermatozoa decreased in both WT and TLR4 mice fed a HFD. The pregnancy rate and litter size declined in the HFD-fed WT mice compared to the HFD-fed TLR4 mice. In conclusion, the HFD alters energy and steroid metabolism in mice, which may lead to male reproductive disorders. However, fertility competence was somewhat restored in HFD-fed TLR4 male mice, suggesting that the TLR4 is involved in testis dysfunction due to metabolic imbalance.

Leiomyosarcoma is malignant mesenchymal tumor of smooth muscle and commonly encountered in the gastrointestinal tract of dogs. However, primary canine leiomyosarcoma in oral cavity is rare due to lack of smooth muscle in the oral tissue. A 13-year-old, neutered male Poodle presented a hard and immobile mass on the left maxilla. Imprinting cytology from the mass as well as fine-needle aspirated cytology from the left scapular lymph node revealed predominant spindle cells met malignancy criteria of the tumors, including coarse chromatin, high N/C ratio, nuclear molding, macro/multi-nucleoli with cigar-shaped nucleus. Radiography of the skull showed lysis of the nasopalatine bone, and mineral radiopacity in the mass. Computed tomography showed soft tissue attenuating mass from the left incisor teeth to the left retrobulbar space with loss of nasopalatine bone and medial wall of orbit. The histopathological examination showed irregularly arranged malignant spindle-shaped cells with oval or elongated nuclei. The nucleolus is distinct and moderate cellular polymorphism is observed. Mitotic figures are occasionally observed. The tumor cells are positive to vimentin, desmin, α-smooth muscle actin when immunohistochemistry was performed, and in Masson’s trichrome stain, tumor cells are stained as red. Overall, histopathologic exam and immunohistochemistry confirmed canine oral leiomyosarcoma. Because of the poor prognosis, the owner did not consent further treatment.

양액 pH가 에케베리아 생육과 색상에 미치는 영향을 알아 보기 위해 본 연구를 수행하였다. 에케베리아 ‘Perle von Nurnberg’ 품종을 선택하여 미국 유타주 프로보(Provo)시 소재 브리검영대학교 Plant and Wildlife 학과의 연구온실에서 수행되었다. 이 품종에 pH 4, pH 7 및 pH 10 3종류의 양액을 공급하여 연구를 수행하였다. 양액 pH 4 처리구에서는 초폭이 120.6mm로 pH 10 처리구의 105.3mm 보다 크게 자랐으며 유의성도 있었다. 하지만 엽장, 엽폭, 엽수에 있어서는 처리간 유의성은 없었다. pH meter를 통해서 식물체 즙액의 pH 를 측정하였는데 4.5에서 4.7 범위의 값을 보였으며 처리간 유의성은 없었다. 위의 결과로 양액의 pH는 식물체의 pH에 영향을 미치지 않는 것으로 판단되었다. 색차계를 이용하여 CIELAB 값을 측정하였을 때 pH 4 처리구에서의 적색도(a)는 -4.0이었으며 pH 10 처리구보다 더 녹색을 띠었다. 명도(L)과 황색도(b) 값은 처리간에 유의적인 차이가 없었다. HPLC를 통한 안토시아니딘 분석에서는 주로 cyanidin, delphinidin 및 pelargonidin의 함량이 모든 처리구에서 상대적으로 높게 나타났다. 공급양액의 pH가 상승함 따라 그 함량도 높아지는 경향을 보였다. Petunidin, malvidin 함량은 미량으로 존재하였으며, peonidin은 분석되지 않았다. 이상의 결과로부터 양액을 pH 10으로 처리하였을 때 에케베리아의 생육억제와 잎 착색에 가장 효과적임을 알 수 있었다.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Classical swine fever virus (CSFV) envelope glycoprotein E2 is the main target for inducing neutralizing antibodies and protective immunity in swine. Here, we report a novel strategy forthe large-scale production of a CSFV E2 subunit vaccine that demonstrates a high immunogenic capability in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (CSFV E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed an approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Mice that were immunized with the granule form of recombinant polyhedra or the soluble form of the fusion protein elicited CSFV E2 antibodies, which indicated that the recombinant polyhedra carrying CSFV E2ΔC were immunogenic. The virus neutralization test showed that the serum from mice that were treated with recombinant polyhedra or the soluble form of the fusion protein contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen and that the recombinant polyhedra containing CSFV E2ΔC as a granule antigen can be used as a potential subunit vaccine against CSFV.

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.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.

Aujeszky’s disease (AD), also called pseudorabies, is an infectious viral disease, caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. AD affects many countries and regions in the world, causing important economic losses, mainly due to international trade restrictions. In this study, to determine the characteristics of the Aujeszky’s disease virus (ADV), NYJ strain, which was isolated from the serum of an infected pig in 1987, we investigated the nucleotide sequence and expression of the glycoproteins gB, gC, and gD using the bBpGOZA system. We found that the glycoproteins gB, gC, and gD of NYJ consisted of 2751 bp, 1443 bp, and 1203 bp, respectively. Comparison of the NYJ with the other strains revealed nucleotide sequence identity ranging from 91.tito 99.0%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The NYJ strain was formed a distinct branch with high bootstrap support. The expression of glycoprotein gD in insect cells was characterized by SDS-PAGE and Western blotting with an anti-ADV polyclonal antibody. Glycoprotein gD of approximately 45 kDa was detected. The results of this study have implications for both the taxonomy of ADV and vaccine development.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

Inflorescence, stem, and leaf samples of lettuce grown in a greenhouse in spring and autumn seasons were assayed for sesquiterpene lactones (SLs) content by high performance liquid chromatography. The concentrations of SLs were significantly higher in the inflorescences followed by upper leaf and stem compared to the other plant parts in most of the samples. SLs content (sum of lactucin and lactucopicrin) in various tissues of lettuce cultivated in spring season varied from 5.7 to 22.5 fold ranging from 27.4 ㎍/g dry weight (DW) in the upper stem (cultivar “PI 176588”) as the lowest to as high as 2,292.0 ㎍/g DW in the inflorescence (cultivar “709849-1”). During autumn cultivation, the concentration of SLs varied from 2.0 to 14.4 fold ranging from as low of 32.4 ㎍/g DW in the lower stem (cultivar “PI176588”) to as high of 838.0 ㎍/g DW in the upper leaf (cultivar “Dambaesangchu”). Higher lactucin (1.2 to 5.6 fold) and lactucopicrin (1.1 to 3.9 fold) concentration was observed during spring compared to autumn cultivation in most of the samples. SLs content in various organs of lettuce increases from the basal plant part going upwards. As lactucin and lactucopicrin are the major SLs which affects the sensory property of lettuce, their quantitative variation in the lettuce cultivars is useful for breeding new varieties with better consumer acceptance.

Background : The purpose of this study is to select potential genetic resources from safflower germplasm collected from India based on their oil compositions and agronomic characteristics. Methods and Results : The agronomic characteristics were measured during the growing period of the safflower. Total oil contents were recovered by Soxhlet extraction and the fatty acid compositions were analyzed by using gas chromatography. The mean of plant height, leaf length and leaf width were 100.19 ㎝, 20.49 ㎝, and 7.29 ㎝, respectively. The percentage of leaf margin with serration was 95%, and 2% of the total resources didn’t have spines on the involucral bract. K185681 had no spines on the involucral bract and the plant height was the smallest. 73% of the flower of safflower was yellow. 68% of safflower germplasm changed flower color from yellow to red. Total oil contents of 267 safflower accessions showed a significant variability among the entire domain of collections and ranged from 5.81 to 38.91%. Palmitic and stearic acid were ranged from 4.98 to 6.65%, and 1.82 to 2.73%, respectively. Oleic and linoleic acid showed a wide variation which ranged from 10.53 to 22.27%, and 69.46 to 81.26%, respectively. Linolenic acid was ranged from 0.06 to 0.13%. K185639 and K185639 had the highest total oil contents and linoleic acid, respectively. Cluster analysis based on oil composition and agronomic characteristics data divided the germplasm collections into three groups. Group Ⅲ having 114 accessions contained accessions with taller plant height than the other groups. Group Ⅱ having 68 accessions, the main color of flower was white but the other groups were yellow. Oleic acid showed a strong negative correlation (r = -0.9691**) with linoleic acid. Principal component analysis (PCA) based on the oil compositions and agronomic characteristics data revealed that the first two principal components (PC1 and PC2) together explained 36.28% total variation. Conclusion : These results showed that K185681, K185639 and K185639 could be useful to develop breeding and functional food.