• Fisheries and Aquatic Sciences
• /
• 제18권4호
• /
• pp.349-357
• /
• 2015

To reutilize fisheries waste, we isolated a bacterial strain from a coastal area located in Busan. It was identified as Bacillus licheniformis TK3-Y. Using plate assay and 500-mL flask experiments, we found that the isolate simultaneously possessed cellulolytic, proteolytic, and lipolytic activities with salt tolerance. 10% (v/v) inoculums, were used to examine the biodegradation characteristics of the TK3-Y strain on carboxymethylcellulose, skim milk, and olive oil media. The optimum conditions for pH, temperature, agitation speed, and NaCl concentration on each 1% substrate were 6, $50^{\circ}C$, 180 rpm, and 17.5%, respectively. Under optimal conditions, the TK3-Y strain showed 1.07 U/mL cellulolytic, 1,426 U/mL proteolytic, and 6.45 U/mL lipolytic activities. Each enzyme was stable within a range of 17.5-35% NaCl. Therefore, the salt tolerance ability of strain TK3-Y was superior to other related strains. In degradation of a mixed medium containing all three substrates, both the cellulolytic and proteolytic activities were somewhat lower than those on each single substrate, while the lipolytic activity was somewhat higher. From the above results, the TK3-Y strain appears to be a good candidate for use in the efficient treatment of fisheries waste in which components are not collected separately.

• 대한화학회지
• /
• 제13권3호
• /
• pp.233-240
• /
• 1969

The synthesis is described of new pepsin substrates of benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine ethyl ester and benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine for studies on the specificity of pepsin, and thin layer chromatographic examination of the peptides prepared showed the new substrates are homogeneous and also, same examination of the incubation mixtures showed that two synthetic substrates are cleaved by pepsin at the L-tyrosyl-L-phenylalanyl bond and hydrolysis of these substrates by pepsin is achieved without transpeptidation. It is found that synthetic peptides are moderately soluble with the amount of the substrate up to a concentration of 0.7 mM in aqueous sodium citrate buffers (0.04 M) in the pH range 1.8-4.0, thus obviating the necessity for the adding of an organic solvent in the assay mixture. The kinetic parameters for synthetic substrates are tabulated in the following table. The data in the table indicate that the susceptibility of synthetic peptides to peptic hydrolysis are relatively large and the change of the carboxyl-terminal group of synthetic substrate from glycine ethyl ester to glycine causes a small decrease in the susceptibility of the L-tyrosyl-L-phenylalanyl bond.

• 생명과학회지
• /
• 제10권2호
• /
• pp.218-227
• /
• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.

• 방사선산업학회지
• /
• 제7권2_3호
• /
• pp.149-154
• /
• 2013

In this study, the simple preparation of poly(sodium 4-styrenesulfonate) (PSS)-patterned substrate via a selective ion irradiation was investigated to manipulate cell adhesion. PSS thin films spin-coated onto the hydrophobic polystyrene (PS) was patterned through masked 150 keV proton irradiation followed by developing with deionized water. The characteristics of the resulting PSS-patterned surfaces were investigated by using microscope, surface profiler, FT-IR, XPS, and contact angle analyzer. These analytical results revealed that the resolved $100{\mu}m$ PSS patterns were formed on the hydrophobic PS surface above the fluence of $1{\times}10^{15}ions\;cm^{-2}$ and the chemical structure, composition, and wettability of the PSS patterns were dependant on a fluence. Moreover, the results of the in-vitro cell culture and proliferation assay exhibited that H1299 cells preferentially adhered and proliferated onto the more hydrophilic PSS part of the PSS-patterned PS and the well-aligned cell patterns was formed on the PSS-patterned PS particularly at the fluence of $1{\times}10^{15}ions\;cm^{-2}$.

• 한국산학기술학회논문지
• /
• 제17권1호
• /
• pp.152-158
• /
• 2016

글루탐산염(Glutamate)은 척추동물의 중추신경계에서 중요한 흥분성 신경전달물질 중의 하나이다. 글루탐산염의 대사를 조절하는 사람 글루탐산염 탈수소 효소(hGDH)는 정신분열증(schizophrenia) 환자의 대뇌에서 발현이 증가한다는 연구들이 있었다. 본 연구에서는 정신분열증과 연관된 항정신성약물인 haloperidol, risperidone, (${\pm}$)-sulpride, chlopromazine hydrochloride, melperone, (${\pm}$)butaclamol, domperidone, clozapine에 의한 hGDH의 효소활성변화를 확인하고자 하였다. 우선, 유전자 재조합을 통해 hGDH 동종효소 hGDH1, hGDH2를 합성하였다. 합성된 hGDH1과 hGDH2에 대한 항정신성약물의 억제효과를 효소검사법(enzyme assay)을 통해 확인한 결과, haloperidol, (${\pm}$)-sulpride, melperone, clozapine에 의해 hGDH1과 hGDH2의 효소활성이 억제되었다. 또한, 단백질 인산화 효소 측정법(kinase assay)을 하여 haloperidol이 기질인 알파-케토글루타르산에 대하여는 비경쟁적 저해반응(noncompetitive inhibition)을, NADH에 대하여서는 반경쟁적 저해반응(uncompetitive inhibition)이 나타는 것을 확인하였다. 입체성 다른 자리 작동체(allosteric effector)인 L-leucine이 다른 정신병치료제에서는 hGDH2의 억제를 회복시켰지만 오직 haloperidol에서는 효소의 활성이 회복되지 않았다. 따라서 본 연구는 hGDH1과 hGDH2 에서 항정신성약물에 의한 효소활성 억제를 비교하여 확인하였으며, 중추신경계에서 haloperidol이 GDH 활성 조절과 함께 글루탐산 농도를 조절할 수 있다는 가능성을 제시한다.

• 한국산림과학회지
• /
• 제86권3호
• /
• pp.261-269
• /
• 1997

미류나무의 미성숙 ovule과 줄기로부터 유기된 캘러스에 plasmid pBI221 유전자를 유전자총을 이용하여 인위적으로 삽입하였다. Plasmid pBI221은 CaMV-35S 유전자에 의하여 발현되는 ${\beta}$-glucuronidase(GUS) reporter 유전자를 포함하고 있다. Plasmid pBI221이 물리적으로 삽입된 후 GUS 유전자의 발현정도는 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-glue)의 반응에 의해 분석되었고, 유전자의 일시적 발현현상은 ovule, 캘러스 시료에 X-glue substrate의 반응에 의하여 나타나는 뚜렷한 점(spot)의 수에 따라서 조사하였다. 본 실험에서 particle bombardment후 GUS유전자의 발현검정에 있어 가장 중요한 요인은 bombardment 횟수와 X-glue substrate에 노출된 시간이었다. X-glue substrate와의 반응결과, 캘러스와 ovule에서 각각 56.8%, 75.9%의 반응 점들을 나타냈다. 여러 처리중 두번의 연속적인 shot와 bombardment 이후, X-glue과 sample을 48시간 반응시킨 후 24시간 동안 alcohol로의 침지가 가장 많은 수의 점을 유기시켰고, 이들 반응으로부터 평균 $25.75{\pm}2.77$(ovule), $11.43{\pm}1.22$(calli)개의 반응 점을 보였다. 유전자총에 의한 외래 유전자 도입에 관한 본 연구는 두종류의 시료로부터 빠른 시간 내에 유전자 발현을 볼 수 있을 뿐만 아니라, 지금까지 Agrobacterium을 이용한 형질전환이 보고되지 않은 미류나무(Populus deltoides)의 형질전환 연구에 대체 방법을 제공하리라 생각된다.

• 한국식품위생안전성학회지
• /
• 제9권3호
• /
• pp.123-131
• /
• 1994

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)

• 대한수의학회지
• /
• 제29권4호
• /
• pp.503-515
• /
• 1989

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to avian infections bronchitis virus(IBV) were standardized. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV Mass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well($100{\mu}l$) and the plates were coated by completey drying at $37^{\circ}C$. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in $100{\mu}l$ volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive(IRP) serum. After repeated titration of IRP and negative serum, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive(S/P) OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; $Log_{10}$ ELISA titer=$5.568({\log}_{10}S/P)+4.161$ Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's.

• Journal of Periodontal and Implant Science
• /
• 제38권sup2호
• /
• pp.299-308
• /
• 2008

Purpose: The aim of this study was to evaluate adhesion and gene expression of the MC3T3-E1 cells cultured on machined titanium surface (MS) and anodized titanium surface (AS) using MTT test, Scanning electron micrograph and cDNA microarray. Materials and Methods: The MTT test assay was used for examining the proliferation of MC3T3-E1 cells, osteoblast like cells from Rat calvaria, on MS and AS for 24 hours and 48 hours. Cell cultures were incubated for 24 hours to evaluate the influence of the substrate geometry on both surfaces using a Scanning Electron Micrograph (SEM). The cDNA microarray Agilent Rat 22K chip was used to monitor expressions of genes. Results: After 24 hours of adhesion, the cell density on AS was higher than MS (p < 0.05). After 48 hours the cell density on both titanium surfaces were similar (p > 0.05). AS had the irregular, rough and porous surface texture. After 48 hours incubation of the MC3T3-E1 cells, connective tissue growth factor (CTGF) was up-regulated on AS than MS (more than 2 fold) and the insulin-like growth factor 1 receptor was down-regulated (more than 2 fold) on AS than MS. Conclusion: Microarray assay at 48 hours after culturing the cells on both surfaces revealed that osteoinductive molecules appeared more prominent on AS, whereas the adhesion molecules on the biomaterial were higher on MS than AS, which will affect the phenotype of the plated cells depending on the surface morphology.

• 한국동물위생학회지
• /
• 제15권1호
• /
• pp.32-45
• /
• 1992

In order to establish and enzyme-linked immunosorbent assay to ILTV, field virus strain of ILTV was propagated in chorioallantoic membrane of the embryonated eggs. purified and used as antigen. The antisera selected from the field samples and immunized chickens based on serum neutralization test were used as the standard positive and negative sera in all tests. It was found that optimal antigen concentration was $2{\mu}g$ of protein per well and a 1 : 100 dilution of standard serum showed low background optical density with negative serum and high P/N values of positive sera. A 1 : 500 dilution of the rabbit anti-chicken IgG peroxidase conjugate produced a high P/N values and thirty minutes was chosen as suitable time to read the optical density of the enzyme substrate reaction and optical density was consistent during the 16 hours after stopper was treated. When coated antigen was kept on microplate for varying time up to 16 hours at $4^{\circ}C$ or $37^{\circ}C,$ no significant difference was observed between the treatment. The coated antigen could be kept without change of antigenicity for at least one month at $-70^{\circ}C,\; -20^{\circ}C,\; 4^{\circ}C$ and room temperature. When blocking buffer contanining bovine serum albumin was mixed directly with conjugate and serum at 10% level induced higher P/N values compared to blocking antigen coated microplate with the same blocking buffer. The coefficience of correlation between ELISA and SN test was 0.577. When antibody response of chickens, vaccinated with ILTV, was examined by ELISA and SN test, antibody rising and decay pattern between the two test was similar until 11 weeks of age. However 12 weeks onward antibody titer checked on by SN test was slightly lower than that tested by ELISA.