Metamorphosis is a development process involving the programmed cell death of obsolete larval organs. Aspartic proteinase cathepsin D (BmCatD) is involved in the silkworm Bombyx mori metamorphosis. Here we show a novel functional role of cysteine proteinase cathepsin B during B. mori metamorphosis. The B. mori cathepsin B (BmCatB) was expressed in the fat body, epidermis, ovary, testis, and hemocyte of the larval and pupal stages. The BmCatB was ecdysoneinduced, expressed in the fat body of the molting, the final larval instar and pupal stages, and its expression led to programmed cell death. RNA interference (RNAi)-mediated BmCatB knock-down inhibited the programmed cell death of larval and pupal fat body, resulting in the arrest of larval-pupal transformation. BmCatB RNAi is up-regulated the expression of BmCatD. Based on these results we concluded that BmCatB is critically involved in the histolysis of the larval and pupal fat body, indicating that BmCatB and BmCatD are mutally regulated during silkworm metamorphosis.

We determined the complete mitochondrial genome (mitogenome) of the jewel beetle, Chrysochroa fulgidissima (Coleoptera: Buprestidae) from two overlapping fragments and subsequent sub fragments. The 15,592-bp long C. fulgidissima mitogenome contains gene arrangement and content identical to the most common arrangement found in insects. Most individual C. fulgidissima mitochondrial (mt) genes were well within the range found in the respective genes of other insects. The 875-bp A+T-rich region is shortest among the coleopteran mitogenomes sequenced in their entirety. The region is interesting in that it contains several stem-and-loop structures and tRNA-like structure found in the A+T-rich regions of other insect mitogenomes. As seen in other insect motogenomes the start codon of C. fulgidissima COI gene also is unusual because no typical start codon is available. Three of the 13 protein-coding genes have incomplete termination codon T or TA. All tRNA formed stable stem-and-loop structure, except for tRNASer(AGN), the DHU arm of which formed a simple loop as seen in many other metazoan mt tRNASer(AGN).

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the geneitself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface asa GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by α3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing α3β4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

Autographa californica 핵다각체병 바이러스(AcNPV)의 다각체 단백질과 초록색 형광 단백질의 융합단백질의 특성을 분석하였다. 초록색 형광 단백질 유전자는 AcNPV의 완전한 다각체 단백질 유전자의 앞쪽과 뒤쪽에 융합하여 다각체 단백질 유전자의 프로모터 조절하에 도입하였다. 이렇게 작성된 재조합 바이러스를 각각 Ac-GFPPOL 또는 Ac-POLGFP이라고 명명하였다. 이들 재조합 바이러스에 의해 감염된 곤충세포주에서는 56kDa의 융합단백질이 발현되었다. 한편, 흥미롭게도 재조합 바이러스 Ac-POLGFP에 의해 감염된 세포주에서는 초록색 형광이 핵내에서만 다각체 유사 granular particle 형태로 관찰되었다. 반면에 Ac-GFPPOP에 의해 감염된 세포도주에서는 대부분 핵내에 존재하였지만, 세포질과 핵 모두에서 초록색 형광을 관찰할 수 있었다. 그러나 발현된 융합단백질은 분명히 다각체단백질을 포함하고 있음에도 다각체는 형성하지 않았다. 이러한 결과들은 융합단백질에서 다각체단백질의 위치와 관련이 있는 것으로 보여진다.

Mushrooms have been widely cultivated and consumed as foods and herbal medicines owing to their various biological properties. However, few studies have evaluated the anti-inflammatory effects of mushrooms. Here, we investigated the effects of mushroom extracts (MEs) on lipopolysaccharide (LPS)-induced inflammation in macrophages (RAW264.7 cells). First, we extracted MEs with either water or ethanol. Using LPS-treated RAW264.7 cells, we measured cell proliferation and NO production. Gene expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 (IL-6), and IL-1β was assessed by RT-PCR, and protein abundance of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) and phosphorylation of p65 were determined by immunoblotting. MEs prepared using both water and ethanol inhibited LPS-induced inflammation in RAW264.7 cells. Nitric oxide (NO) levels induced by LPS were reduced by treatment with MEs. Isaria japonica Yasuda water extracts and Umbilicaria esculenta (Miyoshi) Minks ethanol extracts significantly decreased the mRNA expression of inflammation-related cytokine genes including TNF-α, IL-6, and IL-1β. Similarly, the protein abundance of iNOS and COX-2 was also decreased. The phosphorylation of p65, a subunit of nuclear factor-κB was at least partly suppressed by MEs. This study suggests that mushrooms could be included in the diet to prevent and treat macrophage-related chronic immune diseases.

장 건강에 유익한 프리바이오틱스 소재를 개발하기 위하여 배변을 촉진하는 효능을 지닌 붉은팥을 식품 발효에 이용되는 Bacillus subtilis KCCM 11965P로 발효하여 다음과 같은 결과를 얻었다. 붉은팥의 일반성분은 회분 3.35±0.04%, 조단백질 21.1±0.19%, 조지방 0.35±0.02% 함 유되었다. 붉은팥 원물 1%, 3%, 5%와 Bacillus subtilis KCCM 11965P 3% (v/v)를 접종하여 0, 24, 48, 72시간 배양 하였다. 배양액의 총균수를 측정한 결과 붉은팥 원물을 3% 첨가한 후 72시간 배양군에서 Bacillus subtilis KCCM 11965P 발효가 가장 적합하였다. 발효 시간에 증가함에 따라 총 폴리페놀 함량과 DPPH 라디칼 소거활성이 증가하였다. Protease 활성은 붉은팥 원물 5% 첨가한 후 72시간 배양한 군(2.69±0.003 unit/mL)에서 활성이 가장 높았다. 발효시간과 붉은팥 원물 첨가 농도가 증가함에 따라 α -amylase 활성도 증가하였으며, 붉은팥 원물 5% 첨가한 후 72시간 배양한 군에서 0시간 배양군(1.0±0.1 unit/mL) 보다 26.0±0.2 unit/mL로 증가하였다. Bacillus subtilis KCCM 11965P로 72시간 배양한 후 유리아미노산을 측정한 결과 leucine은 붉은팥 원물 5% 첨가한 0시간 배양군 5.22 mg/L 에서 67.59 mg/L로 증가하였으며, 비필수아미노산인 tyrosine은 5% 첨가 0시간 배양군 10.08 mg/L에서 259.35 mg/L로 증가하였다. 이와 같이 Bacillus subtilis KCCM 11965P로 붉은팥을 발효하면 항산화 활성, protease 효소활성, 및 α-amylase 효소 활성이 증가하였으며, 유리아미노 산과 유기산이 증가하였다. 붉은팥을 발효하는데 Bacillus subtilis KCCM 11965P가 적합할 것으로 판단되며, 붉은팥은 프로바이오틱스를 활성화시켜 장 건강을 증진시킬 수 있는 프리바이오틱스 소재로 개발할 수 있는 가능성을 시사 하였다.

Present study aimed to determine the effect of ‘bitter melon’, a popularly used fruit in Bangladesh and several other Asian countries, on high-fat-diet-induced type 2 diabetes. To investigate the effect, ethanol extract from bitter melon (BME) as a dietary supplement with mouse chow was used. BME was found to significantly attenuate the high-fat diet (HFD) -induced body weight and total fat mass. BME also effectively reduced the insulin resistance induced by the HFD. Furthermore, dietary supplementation of BME was highly effective in increasing insulin sensitivity and reducing hepatic fat and obesity. These results indicate that BME could be effective in attenuating type 2 diabetes and could therefore be a preventive measure against type 2 diabetes.

Background : Recently, hair loss, which has been regarded as a mere means of middle-aged men due to stress and environmental pollution. The market for hair loss in Korea is about four trillion won and it is growing continuously. It is mainly made by mixing natural extracts such as medicinal plant. The purpose of this study was to investigate the effects of ethanol extracts of Houttuynia cordata Thunb. whole plant and Calendula officinalis L. flower extracts on the growth of fibroblasts, dermal papilla cells and lipid precursors, I want to try to make a materialization. Methods and Results : The cytotoxicity of each sample extracts treated with 50%, 100%, and 500 μg to fibroblasts, cell-viability were 107.3%, 109.6%, and 128.2%, respectively. The cytotoxicity of each sample to the dermal papilla cells was not observed. And the lipid differentiation of the lipogenic precursor cells which regulates the hairegeneration by secretion of the platelet derived growth factor. The 70% ethanol extracts of H. cordata whole plant and C. officinalis flower were showed promotes the hair growth activity. The lipolysis rate was significantly increased with increasing treatment concentration Conclusion : As a result of this study, in-vitro hair growth activity of herbal medicines for hair treatment material development was not shown to be toxic to each cell. And 70% ethanol extract of H. cordata whole plant stimulated lipid precursor cells inducing differentiation. As a result, the 70% ethanol extracts of H. cordata whole plant and C. officinalis flower have potential to developing hair-related product.

Steel slag has been used as the alternative binder to replace Portland cement that furthermore used as in construction and/or for stabilization/solidification of heavy metals in mine soil. One of the treatments to modify the leaching behavior of the mine soil is by carbonation. The purpose of this study was to assess the potential of carbonation in various steel slags. Based on chemical and mineralogical characterization of four kinds of slag that were used in this study, it showed that all slags had high potential for reacting with CO2 that was in accordance with the high CaO and MgO content. CO2 sequestration by aqueous carbonation of several kinds of stainless steel slags with different liquid to solid ratio was investigated in this study. The effect of chemical properties and reaction time on the performance of the carbonation process was also investigated. Converter slag, blast furnace slag (BFS) and ladle furnace slag (LFS) were used. Carbonation experiment was conducted in a closed reactor under the conditions; 1bar, 400rpm and 25℃, with solid to liquid ratios of 0.4, 0.6 and 1.0. Carbonation kinetic test was relatively fast and completed within 5 hours. The CO2 consumption increased when the liquid to solid ratio increased because of the dilution effect. Our results showed that the higher CaO and MgO contents in the slag, the higher CO2 consumption was observed. Pohang converter slag and Dangjin LFS showed slightly different tendency. At L/S ratio 0.4, Pohang slag with higher CaO and MgO content had higher CO2 consumption than Dangjin LFS. As the water content increased, Dangjin LFS had higher CO2 consumption than Pohang converter slag that was caused by the texture of Dangjin LFS with smaller particle size than Pohang converter slag. However, both Pohang BFS and Dangjin BFS have poor capacity in CO2 sequestration.

This study aimed to evaluate the role of D-dimer in terminal cancer patients. Among 343 incurable cancer patients, only 4 patients (2.8%) had normal D-dimer levels, and the majority (n=115, 81.0%) had levels >2.0 μg/mL. The survival time was significantly differed according to D-dimer levels with a median of 48 days when D-dimer levels were <2 μg/mL and 19 days when D-dimer levels were higher. D-dimer levels were negatively correlated with survival according to Spearman’s rank correlation analysis (coefficient -0.335, p<0.001). In terminal cancer patients, D-dimer concentrations did exhibit prog-nostic significance.

TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

The innate immune system is the only defense weapon that invertebrates have, and it is the fundamental defense mechanism for fish. The innate immune response is important in newly hatched flounders because it is closely involved in the initial feeding phase, which is why it is essential for survival during the juvenile period. The expression analysis of genes involved in the innate immune response in the olive flounder (Paralichthys olivaceus) in the days after hatching is incomplete. Therefore, we have begun to examine the expression patterns of genes specifically induced during the development of the innate immune system in newly hatched flounders. Microscopic observation showed that pronephron formation corresponded with the expression of perforin-encoding gene. These results suggest that perforin plays a vital role in the innate immunity of the kidney during developmental stages. Perforin expression was strong at the start of the development of the innate immune response, and continued throughout all the development stages. Our findings have important implications with respect to perforin’s biological role and the evolution of the first defense mechanisms in olive flounder. Further studies are required to elucidate the perforin-mediated innate immunity response and to decipher the functional role of perforin in developmental stages.

Fish larvae are immediately exposed to microbes from hatching to maturation of their lymphoid organs, therefore effective innate mechanisms is very important for survival in such an environment. The key component of innate immune system, C3 is central protein of all activation pathways of the complement system, leading to inflammatory reactions, such as opsonisation, chemotaxis, and cell lysis of pathogens. Although, innate mechanisms is essential for survival in the early stage of development, little is known about defence mechanisms. In this study, the alignment analysis showed that amino acid sequence of C3 from olive flounder liver EST homologous to other known C3 sequences with 73-99% identity. Also, we examined the tissue distribution of olive flounder C3 and analyzed expression pattern from the fertilized egg until 28 days post hatching. As a result, olive flounder C3 mRNA was expressed only in the liver and the mRNA level more increased as developmental proceed during the early stage. These results may suggest that olive flounder C3 plays an important function in the early immune response of olive flounder larvae.

Olive flounder (Paralichthys olivaceus) is one of the commercial important flatfish species in Korea. The ocular signal transduction pathway is important in newly hatched flounders because it is closely involved in the initial feeding phase thus essential for survival during the juvenile period. However, the study of gene expression during ocular development is incomplete in olive flounder. Therefore we examined the expression analysis of specifically induced genes during the development of the visual system in newly hatched flounders. We searched ocular development-involved gene in the database of expressed sequence tags (ESTs) from olive flounder eye and this gene similar to arrestin with a partial sequence homology. Microscopic observation of retinal formation corresponded with the time of expression of the arrestin gene in the developmental stage. These results suggest that arrestin plays a vital role in the visual signal transduction pathway of the retina during ocular development. The expression of arrestin was strong in the ocular system during the entirety of the development stages. Our findings regarding arrestin have important implications with respect to its biological role and evolution of G-protein coupled receptor (GPCR) signaling in olive flounder. Further studies are required on the GPCR-mediated signaling pathway and to decipher the functional role of arrestin.

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87－98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

Olive flounder (Paralichthys olivaceus) is a most important aquaculture species in Korea. Like most marine fishes, olive flounders are stomachless at first feeding and aquired gastric function during the metamorphosis, so food was mainly digested by pancreatic enzyme from first feeding to premetamorphosis. However, comprehensive analysis of pancreatic and gastric digestive enzyme of olive flounder at early developmental period is still unclear. In the expression study of pancreatic and gastric digestive enzyme by real-time PCR at early developmental stage, pancreatic enzyme such as chymotrypsinogen 2, preproelastase 2 and 4, pancreatic protein somatomedin-B domain (PPSB) mRNA expression were initiated at first feeding and strongly expressed in the pancreas developmental stage, while gastric digestive enzyme signal was not at all detected during same period. Although, trypsinogens were secreted from pancreas and have similar amino acid sequence, trypsinogen 3 expression induction was detected both pancreas and stomach developmental stage, while trypsinogen 2 expression was significantly increased only post-metamorphosis period. Pepsinogen mRNA expression was only detected at metamorphosis according to stomach differentiation. Lipid digestive enzyme, lipase and intestine fatty acid binding protein 1 (I-FABP 1), were already reached a certain level at beginning of hatching and more increased during early developmental stage and then gradually decreased before metamorphosis. These results suggested that feed ingestion of olive flounder was exclusive charged by pancreatic digestive enyme, lipid digestive enzyme and trypsinogen 3 from first feeding and then fully swiched by gastric digestive enzyme and trypsinogen 2 from metamorphosis period.