The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, SeminarkPro (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at 17℃ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/Dead® stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at 17℃) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90～6.86) and T4 (6.86～6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0～7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 (20×106 sperm cells/ml : 5×105 cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide (H2O2), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decreased with addition of H2O2, and were significantly (p<0.05) lower in medium with 0.1 mM H2O2 than control group. Also, the rate of degenerated oocytes was increased in as H2O2 concentration increased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When H2O2 concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM H2O2 and (3) control medium with 1.0 mM H2O2 along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. H2O2 decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity against oxidative stress caused by H2O2. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM H2O2 alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

The silkworm-baculovirus expression system has distinct advantages, such as a high yield and safe usage in vertebrates. Here, we report a novel strategy for the large-scale production of a classical swine fever virus (CSFV) envelope glycoprotein E2 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 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and 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 contained both the fusion protein and native polyhedrin 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. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedraelicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra 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.

Oleanolic acid is a natural triterpenoid that exists widely in foods and some medicinal herbs. The purpose of this study was to determine the antimicrobial activity of oleanolic acid against Streptococcus mutans strains isolated from a Korean population. Antimicrobial activity against these bacteria was evaluated by minimal inhibitory concentration (MIC) and time kill curves. The tolerance of human gingival fibroblasts and human periodontal ligaments to oleanolic acid was tested using a methyl thiazolyl tetrazolium (MTT) assay. The MIC90 value of oleanolic acid for both S. mutans and S. sobrinus isolated from Koreans was 8μg/mℓ. Oleanolic acid showed bactericidal effects against S. mutans ATCC 25175T and S. sobrinus ATCC 33478T at 1 × MIC(8μg/mℓ) and had no cytotoxic effects against KB cells at this dose. The results suggest that oleanolic acid could be useful in the future development of oral hygiene products for the prevention of dental caries.

Lentinus edodes is a popular edible mushroom in South-East Asia. This study was initiated to evaluate the antioxidant activities, tyrosinase inhibitory effects on the fruiting bodies of L. edodes extracted with acetone, methanol and hot water. The antioxidant activities were performed on β-carotene-linoleic acid, reducing power, 1,1-diphenyl-2-picrylhydrazyl free radical scavenging, and ferrous chelating abilities. In addition to this, phenolic acid and flavonoids contents were also analyzed. Methanolic extract of L. edodes showed the strongest β-carotene-linoleic acid inhibition as compare to others extracts. At 8 mg/ml, hot water extract showed a high reducing power of 0.96. The scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radicals, acetonic extract was effective than other extracts. The strongest chelating effect (86.45%) was obtained from the acetonic extract at 1.0 mg/ml concentration. Antioxidant activities of the extracts from the fruiting bodies of L. edodes were increased with the increasing concentration. After application of reverse phase high performance liquid chromatography, coupled to a diode array detector and electrospray ionisation mass spectra, four phenolic compounds namely, naringenin, hesperetin, formononetin and biochanin were identified from acetonic extract. Tyrosinase inhibition of acetonic, methanolic, and hot water extracts of L. edodes were increased with the increasing of concentration. Results revealed that acetonic and methanolic extracts showed good, while hot water showed moderate activities of the tyrosinase inhibition at the concentration tested. This study suggests that fruiting bodies of L. edodes can potentially be used as a readily accessible source of natural antioxidants.

Lentinus lepideus is an edible mushroom, belongs to the family Tricholomaceteae and order Agaricales. The purpose of this study was to evaluate the antioxidant activities, tyrosinase inhibitory effects on the fruiting bodies of L. lepideus extracted with acetone, methanol and hot water. The antioxidant activities were performed on β-carotene-linoleic acid, reducing power, 1,1-diphenyl-2-picrylhydrazyl free radical scavenging, and ferrous chelating abilities. In addition to this, phenolic acid and flavonoids contents were also analyzed. Hot water extract of L. lepideus showed the strongest β-carotene-linoleic acid inhibition as compare to others extracts. At 8 mg/ml, methanolic extract showed a high reducing power of 1.21. The scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radicals, acetonic and methanolic extracts were effective than hot water extract. The strongest chelating effect (87.50%) was obtained from the methanolic extract at 1.0 mg/ml concentration. Antioxidant activities of the extracts from the fruiting bodies of L. lepideus were increased with the increasing concentration. After application of reverse phase high performance liquid chromatography, coupled to a diode array detector and electrospray ionisation mass spectra, six phenolic compounds namely, chlorogenic acid, vanillin, naringin, naringenin, formononetin and biochanin were identified from acetonic extract. Tyrosinase inhibition of acetonic, methanolic, and hot water extracts of L. lepideus were increased with the increasing of concentration. Results revealed that acetonic and methanolic extract showed good, while hot water showed moderate activities of the tyrosinase inhibition at the concentration tested. This study suggests that fruiting bodies of L. lepideus can potentially be used as a readily accessible source of natural antioxidants.

This study was to investigate the effects of upper and lower limb composing patterns of PNF(proprioceptive neuromuscular facilitation) on the static balance ability by 20 subjects for 6 weeks. This study was measured left one leg standing and right one leg standing with closed eyes on Good Balance system. These results led us to the conclusion that the mean speed of X, Y direction, COP(center of Pressure) velocity moment showed a statistical decrease when applying post-exercise. The above results from this study indicated that upper and lower limb composing patterns of PNF exercise has improved the static balance ability. As a result, this study showed that upper and lower limb composing patterns exercise improve the ability of balance in young adults. Based on this study, it may be applied to old people.

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.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainlyinvolved in the xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTSgene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H2O2 overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H2O2 overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3 h after H2O2 exposure, whereas BiGSTS and BiGSTD are highly induced at 9 h after H2O2 exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitusare differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.

We examined the effect of Bulnesia sarmienti (BS) water extract on hyperlipidemia induced by a high-fat diet. ICR mice were fed a high-fat diet ad libitum for four weeks. Simultaneously, BS water extract was administered intragastrically at 0 mg/kg (control), 30 mg/kg, or 300 mg/kg once daily for four weeks. Male ICR mice were divided into four groups; normal control group (NC), high-fat diet+vehicle treatment group (HF), high-fat diet+BS of 30 mg/kg treatment group (HF+BS30), and high-fat diet+BS of 300 mg/kg treatment group (HF+BS300). The levels of serum biochemical parameters and histological appearances were evaluated. After four weeks, body weight gain and serum levels of triglycerides, total cholesterol, and Low-density lipoprotein (LDL)-cholesterol were significantly higher in the HF group than in the normal control group. Together, serum High-density lipoprotein (HDL)-cholesterol level in the HF group was lower than that in the normal control group. However, treatment with BS resulted in significantly reduced body weight gain and levels of serum triglycerides, total cholesterol, and LDL-choleterol. In addition, serum HDL-cholesterol level in the BS treatment group was significantly elevated, compared to that of the HF group. Histopathological evaluation of the liver showed fat accumulation and swelling of hepatocytes in the high-fat diet group; these abnormalities were ameliorated by treatment with BS. These results suggest that treatment with BS water extract resulted in dose-dependent prevention and mitigation of high-fat diet-induced hyperlipidemia.

Bee venom contains a variety of peptides and enzymes, including serine proteases. Here we describe the molecular cloning and characterization of a serine protease (Bt-VSP) isolated from the venom of the bumblebee Bombus terrestris. The Bt-VSP gene consists of six exons encoding a 358-amino acid protein. The form of Bt-VSP detected in bee venom was the 34-kDa mature protein, which is created by cleavage of the catalytic domain of Bt-proVSP between Arg111 and Val112. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. The finding that Bt-VSP acts as a fibrin(ogen)olytic enzyme is similar to a previous finding that Bi-VSP, a venom serine protease of B. ignitus, exhibits fibrin(ogen)olytic activity. We also compared major venom components in honeybee and bumblebee, and found that bumblebee venom contains a larger amount of serine protease. Furthermore, unlike bumblebee venom, which exhibits fibrin(ogen)olytic activity owing to the presence of a serine protease, it is likely that honeybee venom lacks fibrin(ogen)olytic activity.

Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insects. Recently, apolipophorin-III in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of the apoLp-III in insects. To investigate the genes which have a relationship with apoLp-III in fall webworm larvae, we reduced endogenous Hc apoLp-III mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-III reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-III, which induced an elevated level of superoxide anion in Hyphantria cunea larvae. The observed effect of Hc apoLp-III RNAi suggests that Hc apoLp-III is related to the action/expression of antioxidants, especially MnSOD.

A new insect member of the STAT family of transcription factors (HcSTAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and transcribed in hemocyte, fat body, midgut, epidermis, and Malpighian tubule. Especially, hemocyte and Malpighian tubule showed transcriptional activation of HcSTAT upon Gram-negative and -positive bacteria challenge. Gram-negative and -positive bacteria challenge specifically results in nuclear translocation of HcSTAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vivo treatment with sodium orthovanadatetranslocates HcSTAT to the nucleus in hemocyte cells.

C-Reactive protein (CRP), which is an 118 kDa pentameric protein, was secreted by the liver is an important biomarker for coronary disease, hypertension and inflammation. In this study, a method for CRP detection exploiting quantum dot (Qdot)-antibody conjugate was developed according to an indirect-competitive immunosensing protocol. For this purpose, a streptavidin-bound Qdot605 was linked with a separately prepared biotinylated monoclonal antirat CRP antibody to produce a Qdot-antibody conjugate. The immunosensing was performed at 0.1 and 20 nM of the coating antigen and conjugate, respectively. The current method was found very sensitive in CRP detection, judging from the concentration-dependent fluorescence emission.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that loss of expression of BiPrx1 or BiTPx1 is compensated by up-regulation of expression of the other peroxidase in response to H2O2 overload.

Although whole crop barley are now widely grown as a silage crop in Korea, but silage quality of the whole crop barley produced from farmer's fields have not been published. Therefore, this experiment was conducted to evaluate forage quality of whole crop barley which was participated in Korean quality contest in 2008. These data were classified by region, forage production, added inoculants, planting date and harvest date. Difference on lactic acid of barley silage was detected in the region (p＜0.05), however, there no significant differences in other chemical composition. The moisture and lactic acid were significant differences in dry matter yield of barley silage. There is all the difference between silage added inoculants and control. Differences between planting dates in ash and crude protein (CP) were detected in barley silage (p＜0.05). From comparison within harvest date, lactic acid ㏊d significant differences among barley silage. Differences in forage quality were observed among whole crop barley for silage. Therefore, nutritional quality as well as lactic acid is important in silage quality contest of whole crop barley.

We describe here the cloning and characterization of a cDNA encoding the ferritin heavy chain homologue (TeFerHCH) from the cricket Teleogryllus emma. The TeFerHCH gene spans 1,009 bp and consisted of four introns and five exons coding for 217 amino acids residues. The TeFerHCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region (UTR) of TeFerHCH mRNA. TeFerHCH was grouped with the S type (HCH) in a phylogenetic tree. The TeFerHCH cDNA was expressed as approximately 27 kDa polypeptide in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that TeFerHCH exhibited ubiquitous expression and was upregulated by wounding and iron overload in the fatbody, suggesting a functional role for TeFerHCH in iron metabolism.