• 약학회지
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• 제51권4호
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• pp.253-258
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• 2007

Bombycis corpus, a batryticated silkworm and white-stiff silkworm, is an oriental drug consisting of the dried larva of silkworm, dead and stiffened due to the infection of Beauveria. An peptidyl antimicrobial molecule was purified from B. corpus by reverse phase-column chromatography and HPLC. Its molecular weight was determined to be 2295.45 by using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Its antimicrobial activity was diminished by trypsin digestion. It exhibited a broad antimicrobial spectrum against not only Gram-negative, but also Gram- positive bacteria. Furthermore, it was found to have an antimicrobial activity against vancomycin-resistant enterococci (VRE), methicillin-resistant S. arureus (MRSA), and vancomycin-intermediate resistant S. arureus (VISA). It may be a useful molecule for a new antibiotic development, especially against antibiotic resistant microbe. This substance may play a role in the defense system of this animal against Beauveria bassiana. This is the first report of a peptidyl antimicrobial substance from B. corpus.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.356-358
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• 2003

각질형성세포의 배양법이 개발 후 피부 결손 부위의 치료에 인체 각질형성세포를 배양하여 얻어진 표피를 이식하거나 세포부유물을 도포하는 기존의 방법들은 trypsin 처리 과정을 거치면서 배양된 세포의 부착능력을 가진 단백질이 손상되어 성공적인 피부 재생이 불가능하다. 이 연구에서는 면역결핍 생쥐 모델에서 인간 피부 각질형성에포와 EGF-피브린 고분자 혼합물을 분사하여 피부의 전층 상처를 재생하였다. 조직학 검사와 면역화학검사를 통하여 각질형성세포와 EGF-피브린 고분자를 함께 분사한 경우 이식된 인간 표피세포에 의한 빠른 표피재생을 확인할 수 있었다. 이 피부 재생술은 기존의 배양된 인공피부 sheet의 이식을 이용한 피부재생법에 비해 여러 가지 장점을 가지고 있으므로, 앞으로 화상이나 피부궤양과 같은 피부결손의 효율적인 새로운 치료법으로 사용되어질 수 있을 것이다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.92-94
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• 2003

각질세포의 배양법이 개발 후 피부 결손 부위의 치료에 인체 각질세포를 배양하여 얻어진 표피를 이식하는 방법부터 세포부유물을 도포하는 방법들은 Trypsin 처리 과정을 거치면서배양된 세포의 부착능력을 가진 단백질이 손상되어 성공적인 피부 재생이 불가능하다. 본 실험에서는 이러한 효소처리의 단점을 보완하기 위해서 고분자 미립구에 세포를 효소처리 과정없이 동물실험을 한 결과 21일 후에는 대조군에 비해서 표피가 완벽하게 재생되었고 이식 후 세포의 부착면적을 늘리고 생착율을 높일 수 있었다. 이 연구에서 효소처리 없이 고분자 미립구에서 배양된 세포를 이용한 인공피부 재생이 효과적인 것을 보여주었다. 이러한 모델은 앞으로 화상이나 궤양으로 인한 피부 손실 부위 치료에 사용될 수 있을 것으로 생각된다.

• BMB Reports
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• 제40권2호
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• pp.163-171
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• 2007

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry $\delta$- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, $Trp^{243}$, $Phe^{246}$, $Tyr^{249}$ and $Phe^{264}$, in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at $Tyr^{249}$ and $Phe^{264}$ still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of $Tyr^{249}$ and $Phe^{264}$ within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.

• 대한수의학회지
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• 제29권3호
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• pp.325-331
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• 1989

Five hundred and forty-eight samples of pig heart muscle were collected from the abattoirs of many regions in Korea to reveal the frequency of Sarcocystis infections and to identify the species from June 1988 to April 1989. Heart muscle of the pigs was inspected for sarcocysts by the direct detection technique and for bradyzoites by the trypsin digestion technique. For examination of development of the parasites in the final host, 5 cross bred mature dogs, 5 puppies and 5 kittens were fed 100g, 50g and 50g of the infected meat respectively, four times in 2 days. Of 402 fattened and 146 older culled breeding pigs, 3 fattened pigs and 39 culled pigs were positive for Sarcocystis. Sarcocystis cysts from heart muscle measured an average of $425{\times}169{\mu}m$ and bradyzoites an average of $15.6{\times}3.5{\mu}m$. Of 15 animals, only 2 puppies were infected with Sarcocystis. The prepatent period was 11 to 12 days and patent period was not examined since the puppies were infected with some another infections and one died on day 11 and another died on day 12 after ingestion of the meat. The sporulated oocysts were detected 11 days after ingestion of the meat and sporocysts 12 days from the puppy feces. The sporulated oocysts measured an average of $16.5{\times}11.5{\mu}m$ and sporocysts an average of $12.6{\times}7.9{\mu}m$. On scraping examination of the intestinal mucosa, fully sporulated oocysts were detected in the tip of the intestinal villi. Considering above all descriptions, Sarcocystis in pig heart muscle in Korea was identified with Sarcocystis suicanis.

• BMB Reports
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• 제39권3호
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• pp.270-277
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• 2006

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.131-140
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• 2001

Objective: To know the effects of xenoestrogen on spermatogenesis, we investigated the expression of cytochrome P450s enzymes (CYPscc, $CYP_{17{\alpha}}$, CYP19) and $3{\beta}$-HSD genes involved in steroidogenesis. Methods: Mouse testicular cells were prepared from 15-day-old ICR mice which had only pre-meiotic germ cells by enzyme digestion using collagenase and trypsin. Testicular cells were cultured in DMEM supplemented with FSH (0.1 IU/ml) and 10% FBS or medium with estrogen ($E_2$), bisphenol-A (BPA), octylphenol (OP; $10^{-9},\;10^{-7},\;10^{-6},\;10^{-5},\;10^{-4}M$, respectively) and aroclor 1254 (A1254) known as PCBs for 48 hours. The gene expression of cytochrome P450 enzymes were examined by semi-quantitive RT-PCR. The production of estrogen and testosterone was examined by RIA. Results: As results, expression of CYPscc mRNA was not significantly decreased, but $3{\beta}$-HSD and $CYP_{17{\alpha}}$. mRNA were significantly dose-dependent decreased. And production of testosterone and estrogen were not different except BPA and OP group ($10^{-5}M$). Conclusion: BPA, OP and A1254 might inhibit steroidogenesis by decreasing CYPscc, $3{\beta}$-HSD and $CYP_{17{\alpha}}$. mRNA expression in the mouse testis. These results suggest that BPA, OP and PCBs like as an endocrine disruptors inhibit the productions of steroidogenic enzymes and decrease the production of T and E by negative feedback mechanism. Therefore, these might disrupt steroidogenesis in Leydig cells of testis and would disturb testicular function and subsequently impair spermatogenesis.

• Asian-Australasian Journal of Animal Sciences
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• 제21권2호
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• pp.270-276
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• 2008

The purpose of the present study was to investigate the effects of exogenous non-starch polysaccharides (NSP) enzymes on performance, intestinal content viscosity and digestive enzyme activities of growing pigs fed a rough rice-based diet. A total of 60 crossbred barrows with an initial body weight of 35.16 kg (SD = 0.82) were blocked by body weight and randomly assigned to two treatments with three replications. Each group was fed the diet based on rice with or without exogenous NSP enzymes (2 g/kg of diet). During the 70 days of the feeding trial, all pigs were given free access to feed and water. At the end of the feeding trial, six pigs from each treatment were randomly selected and slaughtered to collect intestinal digesta, intestinal mucosa, and pancreas. The addition of NSP enzymes improved average daily gain (p<0.05) and feed:gain (p<0.05), and decreased viscosity of digesta in the jejunum (p<0.001) and ileum (p<0.01) of pigs. The supplementation of NSP enzymes increased activities of protease (p<0.01), trypsin (p<0.01) and ${\alpha}$-amylase (p<0.05) in duodenal contents. However, digestive enzymes in the pancreas, jejunal and ileal mucosa were unaffected by the supplemental NSP enzymes (p>0.10). The results indicate that the addition of NSP enzymes to rough rice-based diets improved performance of pigs, reduced viscosity and increased digestive activity in the small intestine.

• 한국축산식품학회지
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• 제30권6호
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• pp.923-929
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• 2010

Colostral whey prepared from colostrum (pooled from first six post-partum milkings) was heated for 10 min at $100^{\circ}C$ Heated colostral whey was incubated with 1% enzymes (protein equivalent basis) for 15, 30, 60, 90, and 120 min at $50^{\circ}C$. Papain, pepsin, trypsin, and alcalase produced different degrees of hydrolysis (DH), 10.66%, 12.42%, 10.83%, and 25.31%, respectively, at an incubation time of 120 min. The SDS-PAGE reveals that significant amounts of bovine serum albumin (BSA), ${\beta}$-lactoglobulin (${\beta}$-LG), and ${\alpha}$-lactalbumin (${\alpha}$-LA) survived papain digestion. In contrast, pepsin completely removed BSA but not ${\beta}$-LG present in heated colostral whey. Alcalase completely eliminated BSA, ${\beta}$-LG, and ${\alpha}$-LA. This differential hydrolysis was confirmed by reversed-phase HPLC analysis. Using ion-exchange chromatography, fraction-1 (F-1) was obtained from alcalase hydrolysate at a NaCl gradient concentration of 0.25 M. Reversed-phase HPLC chromatograms of alcalase F-1 showed numerous small peaks, which probably indicate that a variety of new peptides were produced. Iron content of alcalase F-1 was 28.94 ppm, which was the highest among all enzyme fractions, whereas iron content of colostral whey was 36.56 ppm. Main amino acids contained in alcalase F-1 were Thr (15.45%), Glu (14.12%), and Ser (10.39%). Therefore, alcalase can be used to generate good iron-binding peptides in heated colostral whey, and the resulting iron-binding peptides could be suitable as a value-added food ingredient for food supplements.

• Restorative Dentistry and Endodontics
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• 제41권4호
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• pp.283-295
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• 2016

Objectives: In this study, we characterized human dental pulp cells (HDPCs) obtained by different culture methods to establish the most suitable methodology for dental tissue engineering and regenerative endodontic applications. Materials and Methods: HDPCs were isolated by the outgrowth method (HDPCs-OG), the enzymatic digestion method (collagenase/dispase/trypsin, HDPCs-ED), or the combination of both methods (HDPCs-Combined). The expression of mesenchymal stem cell markers (CD105, CD90, and CD73) was investigated. In vitro differentiation capacities of HDPCs into adipogenic, osteogenic, and chondrogenic lineages were compared. Differentiation markers were analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blotting. Results: Our data indicated that whole HDPCs-ED, HPDCs-OG, and HDPCs-Combined could be differentiated into adipogenic, chrondrogenic, and osteogenic cell types. However, we found that the methods for isolating and culturing HDPCs influence the differentiation capacities of cells. HDPCs-OG and HDPCs-ED were preferably differentiated into adipogenic and osteogenic cells, respectively. Differentiation markers shown by RT-PCR and western blotting analysis were mostly upregulated in the treated groups compared with the control groups. Conclusions: Our findings confirmed that cell populations formed by two different culture methods and the combined culture method exhibited different properties. The results of this study could provide an insight into regenerative endodontic treatment using HDPCs.