• 한국동물학회지
• /
• 제28권1호
• /
• pp.31-43
• /
• 1985

$(Ca^{2+}+Mg^{2+})$-ATPase를 쥐의 근소포체로부터 sucrose density gradient centrifugation의 방법을 사용하여 분리 정제하였다. 정제된 효소를 폴리아크릴 아마이드 젤에서 전기영동한 결과, 토끼와 닭의 경우에서와 같이 분자량 115,000인 단일 단백질 띠로 나타났다. 정제된 이 효소의 활설도는 50 $\\muM$의 $Mg^{2+}, Ca^{2+}, Co^{2+}, Fe^{2+}, Min^{2+}$에 의해서는 증가되었고, 같은 농도의 $Zn^{2+}, Cu^{2+}, Hg^{2+}$에 의해서는 감소되었다. Quinine와 quinacrine 같은 antimalarial drug는 이 효소의 활성도에 큰 영향을 주지 않았으나, p-hydroxymercuric benzoate와 phenylmethylsulfonylfluoride는 이 효소의 활성을 억제하였다. 이 효소는 pH 6과 7 사이에서 가장 높은 활성을 나타내었고, ATP를 기질로 사용하였을 때 Km 값은 98 $\\muM$이었다. $(Ca^{2+}+Mg^{2+})$-ATPase는 microsomal fraction에서 선택적으로 분해되었다. $^{3}H-casein$ 이나 ^{125}I-insulin같은 방사성 동위원소로 표지된 기질을 사용하여 단백질 분해에 대한 활성도를 조사해 본 결과, microsomal preparation에 metalloendoprotease가 존재하였다. 그러나 아직까지는 그 효소가 $(Ca^{2+}+Mg^{2+})$-ATPase를 분해하는지는 확실하지 않다.

• 한국동물위생학회지
• /
• 제22권4호
• /
• pp.377-383
• /
• 1999

Polymorphonuclear (PMN) leucocytes are fundamental importance to the body's defense mechanism and play a major role in the local and systemic reactions to infectious disease. Investigation of the physiological and pathological role of the various leucocyte subtypes in host defence mechanisms is dependent upon the isolation of adequate numbers of viable, pure leucocyte fractions. This report describes the separate frequency of PMN leucocytes both from buffy coat layer and from packed RBC layer when bovine peripheral blood was treated with various anti-coagulants such as acid-citrate-dextrose(ACD), ethyldiaminetetraacetic acid(EDTA), sodium citrate and heparin. The separate frequencies of PMN leucocytes from buffy coat layer was 60.4$\pm$9.6%(heparin), 56.8$\pm$11.8%(sodium citrate), 30.6$\pm$14.1%(ACD) and 6.2$\pm$3.7%(EDTA), in order. Those from packed RBC layer monitored with EDTA, ACD, sodium citrate and heparin was 85.0$\pm$4.7%, 84.3$\pm$5.5%, 83.8$\pm$6.5% and 76.3$\pm$7.7%, respectively. The Ficoll-hypaque(FH) density gradient method was used to remove a small part of lymphocytes and/or monocytes from leucocytes in packed RBC layer. With the result that it increased separate frequency of PMN leucocytes from EDTA(89.9$\pm$2.4%), ACD(89.5$\pm$3.6%), and sodium citrate(83.6$\pm$10.3%) than heparin(68.4$\pm$13.9%). These results indicate that the use of EDTA and ACD as anticoagulant Is suitable for the separation of PMN leucocytes from bovine peripheral blood, and that the FH density gradient method is able to increase the separate frequency of PMN leucocytes from packed RBC layer.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• 제23권5호
• /
• pp.452-457
• /
• 2001

Platelet-rich plasma is an autologous source of platelet-derived growth factor and transforming growth factor beta that is obtained by sequestering and concentrating platelets by gradient density centrifugation. We have used platelet-rich plasma for bone graft, especially allobone graft, at implant surgery, sinus lift procedure, and cyst enucleation. This article is retrospective study from October 1999 to November 2000. All cases were good healing and clinical success.

• BMB Reports
• /
• 제28권2호
• /
• pp.129-137
• /
• 1995

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.

• 한국가금학회지
• /
• 제19권1호
• /
• pp.13-16
• /
• 1992

마사츄셋형 전염성 기관지염 바이러스(IBV)를 SPF 발육란의 뇨막강내에서 증식시켜 Sucrose 밀도구배 초원심분리에 의해 정제한 다음 BALB/c 마우스에 면역시켰다. 면역 마우스에서 채취한 비장세포와 마우스 골수암세포와 여러 차례 융합시험을 실시하였다. 많은 융합세포 중에서 IBV에 특이적으로 작용하는 단클론항체(monoclonal antibody ： MCA)를 산생하는 hybridoma클론은 2주밖에 얻지 못했다. 2주의 MCA는 모두 IgG형이었고 IBV중화능이나 혈구응집 억제능이 인정되지 않았다. 간접형광항체법으로 작성된 MCA를 이용하여 인공접종한 닭의 기관도말표본에서 10일간의 시험기간중 계속 IBV를 검출할 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제15권4호
• /
• pp.767-771
• /
• 2005

Expression in yeast may prove more amenable to generating large amounts of viral antigens for a vaccine candidate. We, therefore, cloned the gene encoding the Hepatitis C virus (HCV) structural proteins (C-El-E2, c740) fused in-frame with, and immediately 3' to, the chicken-lysozyme signal peptide (C-SIG) gene and under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. In yeast, the HCV structural proteins were expressed in two different forms: a processed and a nonprocessed aggregated form. Biophysical characterization by sucrose linear gradient centrifugation revealed that both forms were present in the same fractions with a buoyant density of 1.127-1.176 g/$cm^3$. These findings suggest that the efficient synthesis of HCV structural proteins in yeast may be an important tool to study virus assembly and may lead to the development of an HCV vaccine.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 1986년도 추계학술대회
• /
• pp.512.1-512
• /
• 1986

The cdd gene of Bacillus subtilis, encoding the deoxycytidinecytidine deaminase of pyrimidine nucleotide biosynthesis has been cloned into the EcoRl site of pBR322. The recombinant plasmid, pSol, promoted the synthesis of 100-140 fold elevated levels of the enzyme. A comparison of the polypeptides encoded by cdd complementing and non-complementing plasmids in the mini cell showed the gene product to have a molecular mass of approximately 14 kDa. The nucleotide sequence of the gene and 460 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 14337 Da, was deduced to be the coding region for cdd. However, the enzyme has an apparent molecular mass of 54 kDa as determined by gel filteration, whereas sucrose density gradient centrifugation shows 58 kDa. It means that the enzyme could be forming a tetramer in a physiological state. About 28 amino acids of the N-tetramer in a physiological state. About 28 amino acids of the N-terminal presumably form a signal for membrane translocation and six cystein residues are contained in the structure. S1 nuclease mapping indicated that transcription of cdd is initiated 17 base pairs upstream from the translational start. The structural characterization of the odd gene was performed.

• 한국응용곤충학회지
• /
• 제20권2호
• /
• pp.76-82
• /
• 1981

아마리리스에 모자익병을 일으키는 병원바이러스의 기주범위, 물리적성질, 혈청반응을 조사하고 바이러스 입자를 전자현미경으로 관찰을 하였다. 바이러스의 지표식물 검정 결과 C. amaranticolor, V. unguiculata, C. sativus G. globosa둥 21종의 CMV감수성식물에 병징이 나타났다. 바이러스의 내열성은 55C(10분간)이며 내희석성은 $10^{-3}$이고 내보존성은 실온에서 2일이었다. 한천내 확산법을 이용한 항혈청반응결과 뚜렷한 반응대가 형성되었으며, 바이러스흡광도는 260nm에서 최고였으며, 245nm에서 최저였다. 전자현미경에 의한 바이러스입자의 검경결과 직경이 90nm내외인 소구형 입자가 관찰되었다. 이상의 결과를 종합하면 아마리리스에 모자익병을 일으키는 것은 CMV에 의한 것으로 보인다.

• 한국가금학회:학술대회논문집
• /
• 한국가금학회 1999년도 제16차 정기총회및학술발표회
• /
• pp.11-33
• /
• 1999

This study was conducted to determine the embryonic stages for the isolation of the highest number of PGCs and to improve PGCs enrichment method. The primordial germ cells(PGCs) from different sources of chick embryos were isolated. The embryonic stage having the highest number of PGCs from each sources was selected ; 1-day-old embryos for germinal crescent (stage 6-8), 2.5-day-old embryos for blood (stage 17-18) and 5.5-day-old embryos for gonad (stage 27-28). The number of PGCs from one embryonic germinal crescent, blood and gonad was about 87$\pm$1.8, 103$\pm$4.0, and 932$\pm$10.9, respectively. The viability of PGCs after Ficoll from each sources was similar, showing approximately 70%. the PGCs enrichment method was improved using Ficoll density gradient centrifugation. After this step the purity of PGCs from germinal crescent, blood, and gonad was 45$\pm$9.10%, 85$\pm$1.18%, and 86$\pm$0.19%, respectively. Also, PGCs were picked up by mouth pipette to improve the purity. This improved method for the separation of PGCs from different sources will serve as a useful too to preserve the foundation stocks of poultry and to produce germline chimeras.

• BMB Reports
• /
• 제29권4호
• /
• pp.372-379
• /
• 1996

Components I and II (CI&II) are major phosphoproteins in the frog rod outer segments (ROS) of retina, whose phosphorylation is light- and cyclic nucleotide-dependent. Although it was reported that CI & II could be chemically cross-linked to ${\beta}{\gamma}-subunit$ of transducin (${\beta}{\gamma}_t$), it was not clear whether CI&II physically interact with ${\beta}{\gamma}_t$, under native conditions. CI&II extracted by hypotonic washing fo ROS membranes showed an overlapped migration with ${\beta}{\gamma}_t$, in sucrose density gradient centrifugation. The elution profile of CI&II in the peripheral membrane fractions from gel filtration chromatography also overlapped that of ${\beta}{\gamma}_t$. These hydrodynamic parameters indicate that the native molecular state of CI&II in the peripheral membrane fraction appears to be within a complex, most likely with ${\beta}{\gamma}_t$. CI&II coeluted with ${\beta}{\gamma}_t$, showed no phosphorylation by endogenous kinase which phosphorylates a serine of CI&II in other fractions. The purified CI&II were not able to inhibit trypsin-activated cGMP-phosphodiesterase, and CI&II were not recognized by a monoclonal antibody against the ${\gamma}-subunit$ of transducin, indicating that CI&II are not y-subunit of PDE or transducin. Thus, it is likely that native CI&II, which undergo a light-dependent phosphorylation/dephosphorylation cycle, can associate with ${\beta}{\gamma}$, in frog photoreceptor membranes, and the complex formation has an inhibitory effect on the endogenous phosphorylation of CI&II.