• 미생물학회지
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• 제17권3호
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• pp.97-130
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• 1979

Many industrial processes those employ bacteria are subjected to phage infestations. In L-glutamic acid fermentions using acetic acid, the phage infestations of the organisms have been recently recognized. In efforts to elucidate the sources of phage contamination involved in the abnormal fermentation, a series of study was conducted to isolate the phages both from the contents of abnormally fermented tanks and the soil or sewage samples from the surroundings of a fermentation factory, to define major charateristics of the phage isolates, and finally to determine the correlation between the phage isolates and temperate phages originating from the miscellaneous bacterial species isolated from the soil or sewage samples. The results are summarized as follows; 1) All phages were isolated from the irregular fermentation tanks and soil or sewage samples, and they were designated as phage PR-1, PR-2, PR-3, PR-4, PR-5, PR-6, and PR-7, in the order of isolation. These PR-series phages were proved to be highly specific for the variant strains of Br. lactofermentum only, namely, phage PR-1 and PR-2 for Br. lactofermentum No. 468-5 and phage PR-3~PR-7 for Br. lactofemrentum No. 2256. By cross-neutralization test, the 7 phagescould be subdivided into 3 groups, i. e., phage PR-I and PR-2 the first, phage PR-3, PR-4, PR-5, PR-6 the second, and the phage PR-7 the third. 2) The 7 phages were virulent under the experimental conditions. They produced plaques with clear and relatively sharp margins without distinct halo. The mean sizes of plaques were 1.5mm in diameter for phage PR-1 and PR-2, and 1. Omm for phages PR-3~PR-7. Double layer technique modified by Hongo and described by Adams, was applied to assay of the PR-series phages. The factors influencing the plaques were as follows;young age cells of host bacteria cultured for 3-6 hours represented the largest number and size, optimum was pH 7.0, incubation temperature was $30^{\circ}C$, and agar concentration and amount of overlayer medium were 0.6% and 0.2ml, respectively. 3) PR-series phages were stable in 0.05M tris buffer and 0.1M ammonium acetate buffer solution. The addition of $5{\times}10^{-3}M$ magnesium ion effectively increased the stability. Thermostability experiments indicated that PR-series phages were stable at the teinperture between $50^{\circ}{\sim}55^{\circ}C$ in nutrient medium, $45^{\circ}{\sim}50^{\circ}C$ in buffer solution. However, the phages mere completely inactivated at 603C and 65$^{\circ}$C within 10 minutes. The phages were stable at the range of pH6~9 in nutrient medium and of pH 8-9 in buffer solution, respectively. Exposure of the phages to UV for 25, 60 and 100 seconds resulted in the complete loss of infectivily, respectively. 4) Electron microscopy showed that PR-series phage particles exhibited rather similar morphology, differing in the size All of PR-series phages had a multilateral head and had a simple long tiil about three to five times long as compared with head. By the size, phage PR-1 and PR-2, PR-3, PR-4, PR-5, and PR-6 and PR-7 were classified into same groups, respectively. The head and tail size of phage PR-1, PR-5, PR-5(T) and PR-7 were 85nm, 74nm and 235nm and 350mm, and 72nm and 210nm, respectively. 5) Nucleic acids of PR-series phages were double stranded DNA. The G+C contents of phage PR-1, PR-5 and PR-7 were 56.1, 52.9 and 53.7, respectively. The values of G+C contents derived from the $T_m$ were in agreement with the chemically determined values. 6) PR-series phages effectively adsorbed on their host bacteria at the rate of more than 90% during 5 min. K value for phage PR-1, PR-5 and PR-7 were calculated to be $6{\times}10^9 ml$ per minute, respectiveky. The pH of the medium did effect adsorption rate, but both temperature and age of host cells did not. Generally, optimum adsorption condition of phages seemed to be almost same as optimum growth conditions of host bacteria. 7) In one-step growth experiments, the latent periods at $30^{\circ}C$ for PR-1, and PR-7 were about 70, 50 and 55 min, respectively. The corresponding average burst size was 200, 70 and 90, respectively. Lpsis period according to the multiplicity of infection and a phage series. In case of m. o. i. 100, strain No. 2256 (PR-5) and No. 468-5(PR-1) failed to grow and turbidity decreased after 50 and 70min, respectively. 8) In the lysate of a plaque purified phage PR-5 infected bacteria, there observed 2 types ofphage particles, i. e., phage PR-5 and PR-5 (T) of similar morphology but differing at the length of phage tail, and phage tail like particles. The phage taillike particles could be divided into 4 types by the length. Induction experiments of Br. lactofermentum with UV irradiation, mitomycin C or bacitracin treatment produced neither phage PR-5 (T) or phage tail-like particles. 9) No lysis occured when the growth of 7 strains of miscellaneous bacteria, isolated from soil and sewage samples, were inoculated with either phage PR-5 (T) or phage tail-like particles the inoculation of phage PR-5 pellet resulted in the growth inhibition of the orgainsms in the spot test. The lysates obtained from 3 miscellaneous soil derived bacteria following mitomycin C treatment the growth of Br. lactofermentum, but did not lyze the bacterium.

• 생명과학회지
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• 제22권11호
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• pp.1456-1462
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• 2012

소라페닙은 멀티카이네즈 억제제로서 신세포암, 전이성 간세포암 환자의 치료에 효과가 입증된 경구용 항암제이다. 이 연구의 목적은 고속액체크로마토그래피 텐덤질량분석기법(LC/MS/MS)을 이용하여 사람 혈장 내 소라페닙의 농도를 측정하는 효율적인 방법을 개발하고 한국식품의약품안전청(KFDA) 기준에 따라 분석법을 검정하는 것이다. 혈장시료($100{\mu}l$)에 내부표준물질인 chlorantraniliprole을 첨가한 후 이소프로필알콜과 에틸아세테이트로 구성(1:4, v/v)된 0.1% 포름산 함유 추출용액을 혼합하였다. 원심분리 후 상층액을 취하여 원심감압농축하였다. 잔사를 이동상에 재용해하고 Waters사의 역상 XTerra$^{TM}$ C18 칼럼(입자크기 $3.5{\mu}m$)을 장착한 고속액체크로마토그래피 장치에 주입하였다. 액체크로마토그래피는 0.1% 포름산과 10 mM 암모늄 포메이트를 함유한 버퍼용액과 메탄올, 아세토나이트릴을 각각 1:6:3으로 혼합한 용액을 이동상으로 사용하였으며 5분 내에 측정을 완료하였다. 분석대상 물질들은 텐덤질량분석기에서 electrospray 양이온 이온화($ES^+$) 검출방식으로 확인하였으며 소라페닙은 'm/z 465.2 ${\rightarrow}$ 252.5', chlorantraniliprole은 'm/z 484.4 ${\rightarrow}$ 286.2'으로 구성한 multiple reaction monitoring 방법을 사용하였다. 검정 결과, 2-5,000 ng/ml의 농도 구간에서 양호한 직선성($r^2$ > 0.99)과 정확도(90.7-103.9%), 정밀도(10% 이하)를 나타내었다. 새롭게 개발된 LC/MS/MS을 이용한 사람 혈장 내 소라페닙의 농도 측정법은 KFDA 기준을 만족하였으며, 기존의 방법에 비해 민감도가 높은 방법이었다.