Aspergillus niger에 있어서 섬유질 분해효소계의 동질효소 양상에 미치는 기질의 영향

盧載郞,李榮河,鄭宰勳 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

The influence of cellulosic and hemicellulosic substrates on the production of cellulase and xylanase complexes in Aspergillus niger was investigated. The culture conditions with different substrates exhibited profound effects on the level of endoglucanase (CMCase), β-glucosidase, endoxylanase and β-xylosidase, and on their isozyme patterns. However, intracellular and extracellular isozyme patterns of cellulase and xylanase complexes were qualitatively identical and appeared to be simultaneous in the early growth phase. Prolonged incubation led to the increase in the concentrations of isozymes with a little changes in the relative proportions of those isozymes. These results suggest that the biosynthesis of cellulase and xylanase complexes in A. niger is coordinately regulated at the level of induction. Moreover, multiple forms of extracellular cellulase and xylanase complexes seem to be the outcome of specific gene expression and should not be considered solely as the consequence of post-secretional modification of synthesized enzymes.

Inhibition of Mutagenesis and Transformation by Root Extracts of Panax ginseng in vitro

Rhee, Y.H.,Ahn, J.H.,Choe, J.,Kang, K.W.,Joe, C. 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

The root extract of Panax ginseng was investigated for its inhibitory effects on DNA synthesis, mutagenicity, and cellular transformation using V79 and NIH 3T3 cells. DNA synthesis measured by the [^3H]-thymidine incorporation into V79 Chinese hamster lung cells was significantly decreased by the addition of ginseng extract (0-1 ㎍/㎖) to the medium. However, ginseng extract was found to increase the rate of DNA excision repair synthesis in V79 cells in response to treatment with UV radiation or methyl methanesulfonate. The extract also showed decreased mutation frequency when mutagenicity was examined using V79 cells at the hypoxanthing-guanine phosphoribosyl transferase locus as resistance to 6-thioguaning after exposure to methyl methanesulfonate. We also found that the components of ginseng extract continue to exert an inhibitory effect on the transformation of NIH 3T3 cells initiated by 3-methylchloanthrene, methyl methanesulfonate, and 1-methyl-3-nitro-1-nitrosoguanidine.

Avian leucocyte common antigens : molecular weight determination and flow cytometric analysis using new monoclonal antibodies

Chung, Kyeong-Soo,Hyun Soon Lillehoj,Mark Christopher Jenkins 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Chung, K.S., Lillehoj, H.S. and Jenkins, M.C., 1991. Avian leucocyte common antigens: molecular weight determination and flow cytometric analysis using new monoclonal antibodies. Vet. Immunol. Immunopathol., 28: 259-273. New leucocyte common antigens expressed on all the normal chicken leucocytes have been characterized using two monoclonal antibodies designated as K-11 and K-55. These monoclonal antibodies stained virtually 100% of the leucocytes derived from various lymphoid organs including the spleen, thymus, bursa of Fabricius, caecal tonsil and peripheral blood, as well as a monocytic cell line (MC29), aB cell line (LSCC-RP9), and a T cell line (CU12). However, they did not stain mature erythrocytes, intestinal epithelial cells, or chicken embryonic fibroblasts. The two monoclonal antibodies showed different staining patterns and detected non-overlapping epitopes on MC29 cells in two color immunofluorescence analysis. Western blot analysis under non-reducing conditions showed that the monoclonal antibody K-11 recognized three splenic leucocyte proteins with molecular weights of 92, 42 and 41 kDa, whereas the monoclonal antibody K-55 recognized two proteins with molecular weights of 97 and 42 kDa. The data indicate that the monoclonal antibodies K-11 and K-55 recognize novel leucocyte-common antigens which have lower molecular weights than the previously reported leucocyte-common antigen family.

국내분리 Aujeszky's disease virus의 실험적 감염 자돈에 대한 바이러스학적 연구 : Pathogenecity, excretion, distribution and immunogenicity of virus

박정우,전무형,안수환 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

To investigate the etiology, pathogenicity and virological properties of NYJ-1-87 strain of Aujeszky's disease virus (ADV) that was isolated from the diseased piglet in Korea, the virus at 10^(6.0)TCID_50/0.1㎖ was inoculated intranasally and subcutaneously into 30 to 35 days-old piglets. Results obtained through the experiments were summarized as follows. 1. Ten of the infected piglets were clinically observed for 15 days. On the 2nd day post-inoculation(pi), the signs of pyrexia, anorexia and convulsion were noted. On the 4th 7th days pi, nervous signs of incoordination and intermittent spasm were shown in the most of piglets, and one out of 5 piglets infected intranasally was died with severe nervous signs at the 7th day pi. The signs became relieved on the 8th day pi and all of remainder were completely recovered on the 13th to 14th days pi. 2. In hematological study, prominent decrease in the number of total leukocyte and lymphocyte was shown in the ADV-infected piglets on the 6th day pi. On the 8th day pi, the cell numbers were slightly increased and returned to normal level on the 10th day pi. 3. Viral excretion of the ADV-inoculated piglets was examined by swabbing of nasal and oral cavities, and rectal feces. During the periods of the 3rd to11th days pi, the virus was excreted intermittently from nasal and oral cavities, and rectal feces. The nasal excretions were shown the highest virus concentration of 10^(5.2)TCID_50/0.1㎖ at the 5th day pi. 4. Recovery of the inoculated virus from various organs of the piglets that were died or experimentally slaughtered was attempted, and the virus was isolated from the tissues of brain and tonsil by the cultured cell-inoculation method. The highest recovery rate was noted in the tonsil. By indirect immunofluorescence antibody assay using ADV-monoclonal antibody, the viral antigens were detected in tissues of spleen and liver as brain and tonsil on the 7th to 9th days pi. The virus was not isolated from blood and the tissues of lung and kidney throughout the experiments. 5. Titers of virus neutralizing antibody in the piglets experimentally infected with ADV became increased after the 6th to 9th days pi in both of intranasal and subcutaneous inoculation showing the highest titers of 64 to 128 on the 29th day pi. When the antibody levels were measured by radial immunodiffusion enzyme assay, the reactive diameter was enlarged to be positive after the 4th to 6th days pi in both of intranasal and subcutaneous inoculation showing the largest diameter of 13 to 14mm on the 29th day pi.

국내에서 분리된 Aujeszky's Disease Virus의 면역원성 : Ⅰ. Aluminum Hydroxide겔 흡착 불활화 Aujeszky's Disease Virus항원의 면역원성 Ⅰ. Immunogenicity of the Inactivated Aujeszky's Disease Virus with Aluminum Hydroxide Gel Adjuvant

전무형,이헌준,박정우,안수환 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Aujeszky's disease virus(ADV), NYJ-1 strain, isolated from the diseased piglets in Korea was inactivated with binary ethyleneimine or formalin and absorbed in aluminum hydroxide gel. Safety and humoral and cellular immunogenicity of the antigens were investigated in mouse, guinea pig and piglets. In mouse inoculation test, the antigens showed 100% of safety and induced a significant level of ADV antibody. The protective potency following intracerebral challenge with the virulent ADV at 10^7.5TCID_50/0.2㎖ were 60% to 64.3%. In guinea pig inoculation test, it was found that the antigens were quite safe, inducing 63.2 to 124.9 of virus neutralization(VN) titers at the 5th week post inoculation(pi). The protectivity of the antigens against the challenge of the virulent ADV were 62.5% to 75.0%. In piglet inoculation test, the antigens were quite safe after the primary and booster injections, and induced 28.7 to 39.1 of VN titers at the 5th week pi. The high levels of ADV antibodies were also recognized by agar gel precipitation test, latex agglutination test and immunoenzyme assays. Leucocyte adherence inhibition(LAI) test for the piglets revealed 22.3±3.8% to 32.8±6.9% of LAI index at the 4th week pi, and the evident reactions of delayed type hypersensitivity was observed in subcutaneous inoculation of the antigen to the immunized piglets. Following the challenge, the immunized piglets showed none of specific clinical signs of AD and revealed the higher gain of body weight, and the lower secretion of ADV from the nasal cavity as compared with the control group.

受精能獲得 處理法이 소 卵胞卵의 體外受精 및 分割率에 미치는 影響에 관한 硏究

金相根,韓成郁,韓邦根 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

The studies on the carried out to investigate the effects of capacitation method of spermatozoa on the in vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3∼5㎜. The follicular oocytes were cultured in TCM-199 medium containing hormones and FCS for 24∼48hrs in an incubator with 5% CO_2 in air at 38.5℃ and then matured oocytes were again cultured for 12∼18hrs with motile capacitated sperm by preincubation of mKRB, treatment of HIS(high strength ion), Ca-IA(Inophore A), BFF(bovine follicular fluids) and heparin. The results obtained in these experiments were summarized as follows: 1. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution by preincubation of mKRB, treatment of HIS, Ca-IA, BFF and heparin methods were 53.1%, 33.9%, 50.8%, 48.1%, 58.8% and 28.1%, 17.7&, 26.2%, 22.8%, 32.8%, respectively. And the fertilization and cleavage rate of heparin method was of highest of all. 2. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution by both caffeine, BSA and heparin methods were 65.8%, 70.3% and 40.8%, 47.3%, respectively, and those rates were higher treatment of heparin + BSA, heparin + caffeine than treatment of heparin. 3. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with heparin concentration of 2, 5, 10, 20, 40㎍/㎖ were 50.0%, 54.7%, 58.1%, 51.7% and 27.9%, 32.8%, 37.1%, 30.0%, respectively. And the fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with 10㎍/㎖ of heparin was the highest of all. 4. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with caffeine concentration of 10, 20, 30, 40㎍/㎖ were 71.4%, 74.3% and 70.6%, 70.0% and 45.7%, 47.3%, 44.1%, 41.4%, respectively, The fertilization and cleavage rate of spermatozoa fertilized in BO solution with caffeine and heparin together(70.3∼74.3%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%). 5. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with BSA concentration of 5, 10, 20, 30㎍/㎖ were 63.6%, 62.9%, 66.7%, 60.3% and 44.1%, 43.5%, 48.5%, 42.7%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with BSA and heparin together(60.3∼66.73%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%).

백혈병 세포주에 대한 (±)-ar-Turmerone, 자근 및 황금추출물에 의한 항암제의 세포독성 증강효과

이윤영,유관희,김삼용,안병준 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Using the colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide](MTT)assay, we evaluated the chemosensitivity of 8 anticancer drugs{vincristine(VCR), vinblastine(VBL), adriamycin(ADR), cisplatin(CPDD), etoposide(VP-16), cytosine arabinoside(ara-C), bleomycin(Bleo) and cyclophosphamide(CYC)} and the cytotoxicity-enhancing effects of (±)-ar-turmerone and the extracts of the crude drugs {Lithospermum erythrorhizon(LE) and Scutellaria baicalensis(SB)}on the above mentioned anticancer drugs against HL-60 and KG-1 cells among 8 anticancer drugs, VCR, VBL, ADR, and CPDD inhibited the growth of both cell lines by more than 50% while VP-16, ara-C, Bleo, and CYC were less effective. (±)-ar-Turmerone had significant inhibitory effects against both cell lines, showing the ID_50 values of 11.730 ㎍/㎖ and 0.292 ㎍/㎖ for HL-60 and KG-1 cells. respectively. But the extracts of LE and SB roots showed no significant cytotoxic effects. According to ID_50 values, the cytotoxicities of VCR, VBL and ADR against HL-60 were enhanced two, eight and three times by mixing (±)-ar-turmerone, five, seven and three times by adding the extract of LE root, and twenty, six and three times by mixing the extract of SB root, respectively. The cytotoxicities of the above mentioned drugs against KG-1 cell were enhanced two, seven and three times by mixing (±)-ar-turmerone, two, three and three times by conbining wilth the extract of LB root, and two, five and two times by adding the extract of SB root, respectively. The cytotoxicity-potentiating effects of (±)-ar-turmerone and the extracts of LE and SB roots against HL-60 cell were greater than KG-1 cell.

Podophyllotoxin Analogs : Effects on DNA Topoisomerase Ⅱ, Tubulin Polymerization, Human Tumor KB Cells, and Their VP-16-Resistant Variants

LIU, SU-YING,HWANG, BYUNG-DOO,HARUNA, MITSUMASA,IMAKURA, YASUHIRO,LEE, KUO-HSIUNG,CHENG, YUNG-CHI 충남대학교부설 생명공학연구소 1992 생물공학연구지 Vol.2 No.-

Several derivatives of podophyllotoxin with modifications at the C-4 position of ring C, in addition to demethylation at the C-4' position of ring E, were examined for inhibitory activity against DNA topoisomerase Ⅱ and tubulin polymerization, generation of protein-linked DNA breaks, and cytotoxicity against KB cells and VP-16-resistant KB variants. Substitution of podophyllotoxin with a group in the β configuration at the C-4 position of ring C resulted in compounds with greater inhibitory activity against DNA topoisomerase Ⅱ and lower inhibitory activity against tubulin polymerization than those with an α configuration. These active analogs exhibited the same mechanism of DNA topoisomerase Ⅱ inhibition as the epipodophyllotoxin derivative VP-16, which causes protein-linked DNA breaks in vitro as well as in cells. Two analogs selectively inhibited DNA topoisomerases Ⅱ to a greater extent than tubulin polymerization. These analogs were cytotoxic towards KB cells in addition to VP-16-resistant KB cell lines, which indicated limited cross-resistance with VP-16 in VP-16-resistant KB variants.

급성 림프구성 백혈병 환아의 말초혈액 T-림프구 아형과 림프구 증식능의 의의에 관한 연구

변상현,서종진,정용헌 충남대학교부설 생명공학연구소 1991 생물공학연구지 Vol.1 No.-

To evaluate the relationship between peripheral blood T-lymphocyte subsets, mitogen-induced lymphocyte proliferation and the outcome of acute lymphoblastic leukemia in childhood, the authors studied 15 cases of acute lymphoblastic leukemia in childhood. The results were as follows: 1) Percent peripheral lymphocyte and percent CD8+ cells showed no significant difference between total patient group and control group, while percent CD4+ cells was significantly decreased in total patient group compared to control group. But no significant difference was found between patient subgroups. 2) Total counts of CD4+ cells and CD8+ cells showed no significant difference between patient subgroups. 3) The ratio of CD4+ cells and CD8+ cells showed no significant difference between patient subgroups, but more cases with the ratio less than 1.0 were found among total patient group compared to control group. 4) The stimulation indices of PHA and Con-A were significantly decreased in total patient group (p<0.05, p<0.005) compared to control group, but no significant difference was observed between patient subgroups. 5) The unstimulated ^3[H]-thymidine uptake showed no significant relationship between patient subgroups. 6) The distribution of T-lymphocyte subsets showed no significant relationship with the stimulation index of PHA and Con-A. These results showed that the children with acute lymphoblastic leukemia have depressed cellular immune functions tested with T-lymphocyte subsets and mitogen-induced lymphocyte proliferation assay. But these results can not be regarded as one of prognostic factors of acute lymphoblastic leukemia in childhood unless there are additional longterm data indicating the T-cell mediated immune functions are related to the outcome of acute lymphoblastic leukemia in childhood.

급성골수성백혈병에서의 시험관내 집락세포 형성에 관한 연구

고석만,김종완,박철신,조덕연,김상용,노흥규 충남대학교부설 생명공학연구소 1991 생물공학연구지 Vol.1 No.-

To evaluate the in vitro granulocyte-macrophage colony formation in acute myeloblastic leukemia and the prognostic implications of these results, the author performed the in vitro agar culture of bone marrow cells in 15 patients with acute myeloblastic leukemia(AML) and 5 control subjects. Culture medium was composed of 20% fetal calf serum(FCS), 50% Iscove's medium, 0.3% agar, 10% colony stimulation factor(CSF), and 2×10 exp (5) cells/㎖. Human placental conditioned medium(HPCM) and phytohemagglutinin-leukocyte conditioned medium(PHA-LCM) were used as coloy stimulating factor. Colony counting was done on 7th day of culture. Colony was defined as containing 20 or more cells, and cluster was defined as containing 3-19 cells. The results were as followings, 1. In control subjects, the number of clusters formed was 3-47/2×10 exp (5) cells(20±19) and that of colonies was 5-24/2×10 exp (5) cells(14±9) when stimulated with HPCM. When stimulated with PHA-LCM, the number of clusters formed was 5-39/2×10 exp (5) cells(18±6) and that of colonies was 6-13/2×10 exp (5) cells(9±3). 2. In AML patients, 3 groups were recognized according to pattern of colony formation : 1) non-forming, 2) cluster forming, 3) both cluster and colony forming. Of the 15 cases, 10 cases were 'non-forming', 2 cases were 'cluster forming,' and 3 cases were 'cluster and colony forming'. 3. 8 of 10 cases of 'non-forming' cases, one of 2 cases of 'cluster forming,' and none of 3 cases of 'cluster and colony forming' achieved complete remission. So, there was significant difference in remission rate in the different growth types. These results suggest that granulopoiesis in AML patients is impaired and the pattern of in vitro CFU-L(colony forming unit-leukemia) formation has prognostic significance.