Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putativelow-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identifiedfrom honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated.In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shownto act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-likedomain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putativelow-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide.Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin,but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however,it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPIinhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial andfungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. Thesefindings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

To determine the effect of the tumor necrosis factor-α (TNF-α) in odontoclast formation, we administrated a TNF-α inhibitor in rats with diabetes rats with periodontitis. The rats included in the study were divided into three groups: control rats without diabetes or periodontitis (the C group), rats with periodontitis and diabetes (the PD group), and rats with periodontitis and diabetes treated by infliximab, the TNF inhibitor (the PD+infliximab group). The PD and PD+ infliximab groups received intravenous administrations of streptozotocin (STZ, 50 mg/kg) to induce diabetes. After 7 days of STZ injections, the mandibular first molars were ligatured to induce periodontitis. The PD+infliximab group was intrapenitoneally administrated by infliximab (5 mg/kg). On days 3 and 20 after the ligature administration, odontoclast formation along root surfaces was evaluated by tartrate resistant acid phosphatase (TRAP) staining and cathepsin K immunohistochemistry. On day 3, the number of TRAP- and cathepsin K-positive cells increased more so in the PD group than in the C group. The PD+infliximab group showed a lower number of positive cells than the PD group. There was no difference in all the groups on day 20. On day 3, the cathepsin-K positive multinucleated and mononucleated cells were higher in the PD group than in the C group. The number of cathepsin-K positive multinucleated cells was lower in the PD+infliximab group than in the PD group. The PD group showed more cathepsin K-positive cells in the furcation and distal surfaces than the c group. The Cathepsin K-positive cells of the PD+infliximab group were lower than that of the PD group in furcation. These results suggest that TNF-α stimulates odontoclast formation in diabetes with periodontitis.

The glucosidase inhibitors are currently interested owing to their promising therapeutic potential in the treatment of disorders such as diabetes, human immunodeficiency virus (HIV) infection, metastatic cancer, and lysosomal storage diseases. Among them, β-d-glucosidase inhibitors can be used to elucidate the mechanisms of various biochemical reactions and to treat the Gaucher disease as potential therapeutics. In this study, the natural β-glucosidase inhibitor was isolated from jujube leaf extract. The isolation and purification are vital for the structure analysis. The jujube leaf was extracted with hot water (95°C, 15 min). Then the natural β-glucosidase inhibitor was isolated using size exclusion chromatography and semi-preparative HPLC. In the first purification process, the 35 fractions were collected according to molecular size using PD-10 column packed with Sephadex G-25 medium. The strongest inhibition on the activity of β -glucosidase was observed at 6 and 7 fractions. The chromatogram of these samples showed that 3 peaks were observed comparing with baseline. For further purification, the strongest inhibitory activity peak was isolated using semi-preparative HPLC with VDS C18 column. Highly purified inhibitor, which can be used for structure analysis and characterization, was obtained. Further study is necessary to identify the natural β-glucosidase inhibitor in the jujube leaf extract.

Serine protease inhibitors play a critical role in physiological processes and immune responses by regulating serine protease activities. Here we describe the molecular cloning and antimicrobial activities of a serine protease inhibitor from the mason bee, Osmia cornifrons (OcSPI). OcSPI consists of 405 amino acid residues and contains a potential reactive center loop (RCL) region in its C-terminus. Recombinant OcSPI was produced as a 64-kDa glycoprotein in baculovirus-infected insect cells and exhibited inhibitory activity against chymotrypsin. Additionally, OcSPI demonstrated inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, but not against tissue plasminogen activator, thrombin, or plasmin. Recombinant OcSPI bound directly to Escherichia coli, Bacillus subtilis, and Beauveria bassiana and exhibited antimicrobial activity against both bacteria and fungi. Our results demonstrated the antimicrobial functions of OcSPI and suggest a role for OcSPI in the immune response of O. cornifrons.

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee (Bombus ignitus) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana, Ascosphaera apis, and Fusarium graminearum, but they did not bind to Escherichia coli, Paenibacillus larvae, or Bacillus thuringiensis. Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide.

The cloning efficiency is extremely low despite successful somatic cell nuclear transfer (SCNT) method producing cloned animals in several mammals. In general, faulty epigenetic modifications underlying the incomplete reprogramming of donor cell nuclei after SCNT mainly results in low cloning efficiency. The nuclear reprogramming process involves epigenetic modifications, such as DNA demethylation and histone acetylation, which may be an important factor in enhancing the cloning efficiency. Recently, the histone deacetylase inhibitors (HDACi), such as trichosatin A (TSA) and m-carboxycinnamic acid bishydroxamide (CBHA), to increase histone acetylation have been used to improve the developmental competence of SCNT embryos. Therefore, we compared the effects of TSA with CBHA on the in vitro developmental competence and pluripotency-related gene expression (Oct4, Nanog and Sox2) in porcine cloned blastocysts and histone acetylation pattern (H3K9ac). The porcine cloned embryos were treated with a 50nM concentration of TSA and 100μM concentration of CBHA during the in vitro early culture (10h) after cell fusion and then were assessed to cleavage rate, development to the blastocyst stage and pluripotency-related gene expression in NT blastocyst also, level of histone acetylation in zygote, 2cell, 4cell stage. As results, Although NT, TSA and CBHA treated NT embryos were not different between all groups for cleavage rates, the developmental competence to the blastocyst stage was significantly increased in CBHA treated embryos (22.7%) compared to that of normal NT and TSA treated NT embryos (8.1% and 15.4%)(p<0.05). In addition, all of pluripotent transcription factors (Oct4, Nanog and Sox2) were expressed in the CBHA treated NT embryos, however, Sox2 and Oct4 were expressed in TSA treated NT embryos and expression pattern of CBHA treated NT embryos is particularly similar to that of IVF embryos. Also, CBHA treated NT embryos were increased in level of histone acetylation (H3K9ac) at the zygote, 2-cell, 4-cell stage compared to those of NT and TSA treated NT embryos. In conclusion, the treatment of CBHA as a histone deacetylase inhibitor significantly increased the developmental competence of porcine NT embryos and pluripotency-related gene expressions(Oct4, Nanog and Sox2) in NT blastocysts and level of histone acetylation (H3K9ac).

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a 15 μM of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.

Cotesia plutellae, an endoparasitoids braconid wasp, possesses a polydnaviruses (PDVs) called Cotesia plutellae bracovirus (CpBV) that encodes viral histone H4 (= CpBV-H4). This viral histone H4 shares high sequence homology (82.5%) with host`s H4 of P.xylostella, except an extended N-terminal tail consisting of 38 amino acid residues with nine lysines. Its extended N-terminal tail has been postulated to play a crucial role in suppressing host immunity, growth and development-associated genes, presumably through an epigenetic control mechanism. A suppression subtractive hybridization (SSH) analysis was analyzed in transcriptome by short-read sequencing technology and provided several target and non-target genes of a viral histone H4. In this study, we analyzed the effect CpBV-H4 on the expression of two target genes: Lysine-specific demethylase (KDM) and Serine proteinase inhibitor (Serpins). Transient expression of CpBV-H4 into non parasitized P. xylostella was performed by microinjection of a recombinant expression vector, and showed the expression up to 70 h. Under this transient expression condition, we analyzed the effect of CpBV-H4 on the expression of target genes by RT-PCR at different time points. Interestingly, the CpBV-H4 significantly inhibited the expression of these target genes, while the truncated CpBV-H4 deleting the N-terminal tail did not show this inhibitory effect. This study also showed that the viral histone H4 suppresses expressions of lysine-specific demethylase and serine proteinase inhibitor (Serpin2) to inhibit host growth and development.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine em-bryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in 5 μM treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were sig-nificantly decreased in the 5 μM E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from 5 μM E-64 treated and non-treated groups. Expre-ssion of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expre-ssion of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

1-deoxynojirimycin (1-DNJ), a potent a-glycosidase inhibitor, has therapeutic applications in treatments of HIV, Gaucher’s disease, and diabetes. 1-DNJ has been extracted from natural sources (mulberry leaves) for therapeutic purposes; however, 1-DNJ ingredients are in limited supply and are costly to obtain on a large scale. Since certain strains of Bacillus and Streptomyces species reportedly produce 1-DNJ, they may serve as potential sources for high-yield 1-DNJ production. In this study, we obtained evidence for four bacteria that produce 1-DNJ in large quantities by high performance liquid chromatography and thin layer chromatography. Investigation of the effect of mulberry leaves powder concentration(1~5%), using the 1-DNJ high-production bacteria, provided evidence for microbial mass production of 1-DNJ.

Bumblebee venom serine protease inhibitors have been shown to inhibit plasmin activity. In this study, a bumblebee (Bombus ignitus) venom serine protease inhibitor (BiVSPI) that acts as an antimicrobial factor was identified. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI was expressed in the venom gland and was present as an 8-kDa peptide in venom. Recombinant BiVSPI expressed in baculovirusinfected insect cells exhibited inhibitory activity against chymotrypsin, but not trypsin. BiVSPI also exhibited inhibitory activity against microbial serine proteases, such as subtilisin A (Ki 6.57 nM) and proteinase K (Ki 7.11 nM), indicating that BiVSPI acts as a microbial serine protease inhibitor. In addition, BiVSPI was also shown to bind directly to Bacillus subtilis, B. thuringiensis, and Beauveria bassiana, but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide novel evidence for the antimicrobial function of this bumblebee venom serine protease inhibitor.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or typsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, B. thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.

Lipoxygenase(LOX)와 kunitz trypsin inhibitor(KTI) 단백질이 결핍된 non-GM콩(개척#2, 진양콩 및 CJ#1)의 가공적성을 알아보기 위하여 일반콩(태광콩)을 대조로 하여 간장을 제조하여 이화학적 특성 및 항산화 활성을 비교하였다. 원료콩의 일반성분은 시료간에 비슷한 조성을 보였으나, 총 페놀 및 플라보노이드 함량은 태광콩에 비해 LOX와 KTI 단백질 결핍콩에서 유의적으로 높았다. 간장 중 총당 및 환원당 함량은 태광콩 간장이 가장 높았으며, 총 질소 및 아미노태 질소 함량은 진양콩 간장이 가장 높았다. 무기물은 진양콩 및 CJ#1 간장, 아미노산은 3종의 LOX 및 KTI 단백질 결핍콩 간장이 태광콩 간장에 비해 높은 함량이었다. 간장의 갈색도는 진양콩 간장이 가장 높았으며, 황색도는 진양콩 간장이 태광콩 간장과 비슷한 수준이었다. 간장의 종합적인 기호도는 단맛과 구수한 맛이 높았던 진양콩 간장의 점수가 가장 높았다. 간장의 총 페놀 및 플라보노이드 함량은 LOX와 KTI 단백질 결핍콩 간장이 태광콩 간장에 비해 유의적으로 높았다. ABTS 및 DPPH 라디칼 소거활성은 총 페놀 함량에 의존적이었으며, FRAP법에 의한 환원력은 개척#2 및 CJ#1 간장이 태광콩에 비해 유의적으로 높았으며, Fe2+ 킬레이트 활성은 태광콩 간장이 여타 간장의 활성에 비해 높았으나, 진양콩 간장과 비슷한 수준이었다. 따라서 LOX와 KTI 단백질 결핍콩으로 제조한 간장은 일반콩 간장에 비해 영양적 성분 및 항산화 활성이 높아 기능성 가공식품 소재로써 활용 가능성이 있는 것으로 판단된다.