A simple method for the identification and assay of extracellular plant beta-galactosidase

A simple, rapid and reproducible procedure for the identification of extracellular Californian poppy (Eschscholzia californica Cham.) beta-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-days-old seedlings of Californian poppy germinating on agar plates and cell suspension cultures cultivated from callus cultures. 6-Bromo-2-naphthyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-galactopyranoside were used as substrates for the determination of the intracellular and extracellular activities of beta-galactosidase. The extracellular beta-galactosidase activity was identified by evaluating the dye-zones in an agar medium. The enzyme from Californian poppy callus cultures or from seedling roots cultivated on agar plates supplemented with 6-bromo-2-naphthyl-galactopyranoside hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding (reddish-brown) azo-dye was formed. The agar plate method described permits rapid, simple and specific detection of plant producers of extracellular beta-galactosidase.     

Cephalosporin C acylase in the autolysis of filamentous fungi

Cephalosporin C acylase activity was studied using fluorescamine determination of free--NH2 groups produced in the deacylation of cephalosporin C by the enzyme. Fourteen fungi from different genera were studied and low extracellular cephalosporin C acylase activity was found in the genera Aspergillus, Fusarium and Penicillium. Forty one fungi of these genera were checked but not all presented acylase activity. The enzyme was generally found to be an extracellular enzyme and during the process of autolysis its activity increased with incubation time and with increasing pH of the medium. In no case was beta-lactamase activity detected. Penicillium rugulosum and Penicillium griseofulvum were identified as good cephalosporin C acylase producers. Deacetyl esterase activity was also detected in these fungi.     

Phosphate Mediated Regulation of Cinnamoyl Putrescine Biosynthesis in Cell Suspension Cultures of Nicotiana tabacum

The accumulation of cinnamoyl putrescines by cell suspension cultures of NICOTIANA TABACUM was enhanced manifold by phosphate limitation of the culture medium while growth was reduced under such conditions. The enhanced product formation was preceded by a large increase and subsequent decline of phenylalanine ammonia lyase activity and a smaller increase of the activity of ornithine decarboxylase. Phosphate concentrations commonly used in cell culture media suppressed the product accumulation.     

Biotransformation of cholesterol to diosgenin by freely suspended and immobilised cells of Dioscorea bulbifera L

The cell suspension cultures, established from the friable callus which was risen from the nodal segments of Dioscorea bulbifera L. in Murashige-Skoog (MS) medium supplemented with indole-3-butryic acid (20 mg L(-1)), was examined for cell growth in MS medium fed with cholesterol (50 mg L(-1) and 100 mg L(-1)) after 8, 10, 12, 14, 16, and 18 days of culture. The growth index of the cell suspension culture on the 8th day was 1.2 and gradually inclined to 1.9 on the 16th day and remained the same at the 18th day. There is no marked difference in the cell growth of cholesterol-treated and control cell suspension culture. The maximum accumulation of diosgenin was noticed on the 14th day in control and cholesterol-treated cell suspension culture and immobilised cell cultures. The highest concentration of diosgenin, 2.94% and 2.14% dry weight, was obtained in immobilised cell culture and cell suspension culture treated with 100 mg L(-1) cholesterol, respectively.     

白花蛇舌草悬浮细胞分批培养规律的研究

目的利用白花蛇舌草茎尖的分生组织建立植物悬浮细胞培养系,确定白花蛇舌草悬浮细胞分批培养时的变化规律。方法通过接种不同量的细胞液确定最适接种量,以细胞干重、蔗糖、铵离子、硝酸盐氮和多糖含量作为检测指标,确定白花蛇舌草悬浮细胞分批培养时的变化规律。结果白花蛇舌草悬浮细胞分批培养的最适接种量为15%,此时细胞干重达到最大值。在此接种量下,当培养时间达到9 d时,pH很快降低到3左右,细胞干重不再增加。培养液中的主要营养成分碳源———蔗糖,在培养开始时,有比较大的降低,在培养后期,细胞干重不再增加时,也不再有大的改变。氮源[NO3-]优于[NH4+]先被利用,而且[NO3-]的利用速率要远高于[NH4+],达到7.14μg/(mL.d);细胞液中多糖的生成和细胞的生长属于非偶联型,在培养后期,逐渐大量生成。结论初步确定白花蛇舌草悬浮细胞分批培养时的变化规律,为以后的培养工艺优化打下了基础。     

Regulation of heme metabolism and cytochrome P-450 levels in primary culture of rat hepatocytes in a defined medium

Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of delta-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30-40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19-24 hr when its activity was about eight times the original activity. In 2-4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of delta-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 microM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme.     

Comparative study of the effects of various culture conditions on cell growth and Gagaminine synthesis in suspension culture of Cynanchum wilfordii (MAXIM.) HEMSLEY

Gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordii, exhibited potent inhibitory effects on aldehyde oxidase activity and lipid peroxidation. To determine whether it would be possible to mass produce this active component, which would be useful for animal tests, we tried to synthesize it using in vitro cell culture methods with various growth conditions. In a previous study it was found that calli were easily induced from the stem of this medicinal plant and cultivated effectively on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/l. In this work we attempted to determine the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture. Gagaminine production was increased markedly when cell growth proceeded to the death phase. Cell growth was more effective with 5% (v/v) sucrose, in the light (at 38 microE/m(2) x s), on medium containing 2,4-D 2 mg/l, with 2.5 g/10 ml medium as the initial cell concentration. The concentration of gagaminine was optimal with 3% sucrose, in darkness on medium 2,4-D 1 mg/l, with 2.5 g/10 ml medium as an initial cell concentration. However, the highest growth rate was 0.18 d(-1), when the gagaminine concentration was seven- and three-fold (at 140 mu/ml) that of the plant stem and 10 ml of medium respectively, on the 50 ml of medium in suspension culture.     

Biotransformation of cholesterol to diosgenin by freely suspended and immobilised cells of Dioscorea bulbifera L.

The cell suspension cultures, established from the friable callus which was risen from the nodal segments of Dioscorea bulbifera L. in Murashige-Skoog (MS) medium supplemented with indole-3-butryic acid (20 mg L^- 1), was examined for cell growth in MS medium fed with cholesterol (50 mg L^- 1 and 100 mg L^- 1) after 8, 10, 12, 14, 16, and 18 days of culture. The growth index of the cell suspension culture on the 8th day was 1.2 and gradually inclined to 1.9 on the 16th day and remained the same at the 18th day. There is no marked difference in the cell growth of cholesterol-treated and control cell suspension culture. The maximum accumulation of diosgenin was noticed on the 14th day in control and cholesterol-treated cell suspension culture and immobilised cell cultures. The highest concentration of diosgenin, 2.94% and 2.14% dry weight, was obtained in immobilised cell culture and cell suspension culture treated with 100 mg L^- 1 cholesterol, respectively.     

生孢梭菌生长所需最低水分活度研究

目的探讨用不同介质调节培养基的水活度,严格厌氧微生物生孢梭菌生长所需的最低水分活度。方法用氯化钠、蔗糖及甘油调节梭菌增菌培养基的水分活度,取制备好的新鲜菌悬液,用无菌生理盐水稀释成每1 mL含104 cfu的菌悬液。将上述菌液分别加入3种调节好的梭菌增菌培养基水分活度中,取1 mL稀释至适宜的稀释级注平皿倾注哥伦比亚琼脂培养基,待培养基凝固后装入厌氧培养袋,在35℃培养72 h,计数。结果以氯化钠、蔗糖及甘油调节梭菌增菌液的水分活度,生孢梭菌生长所需最低水分活度分别为0.95~0.96,0.95~0.96,0.92,其中甘油的试验结果相对稳定,生孢梭菌生长所需最低水分活度最低。结论该方法可为水分活度测定在我国非无菌制剂微生物控制中的应用和2020年版《中国药典》相关内容的修订提供参考。     

Enhanced resistance of HeLa cells to cisplatin by overexpression of gamma-glutamyltransferase

Gamma-glutamyltransferase (GGT), which is a key enzyme for the cellular glutathione (GSH) homeostasis, was shown to be overexpressed in human tumor cells selected for resistance to cisplatin and to influence the resistance of experimental tumors in vivo. We first established that cisplatin treatment of HeLa cells was accompanied by an early 3-fold induction of GGT synthesis, enhancing the possibility that this enzyme plays an important role in the cell defenses against this anticancer drug. This role was then studied using a GGT-transfected HeLa cell line (HeLa-GGT) exhibiting 10 times the activity of the parental HeLa cells (120-150 and 10-14 mU/mg protein, respectively). Both cell lines showed comparable intracellular GSH levels and cisplatin resistance when cultured in high (250 microM) or low (50 microM) cysteine-containing medium. When 50 microM of GSH were included in the low-cysteine culture medium only HeLa-GGT cells partially recovered their intracellular GSH and exhibited an increased resistance to cisplatin. Cisplatin treatment also inhibited GGT-dependent production of reactive oxygen species, a process depending on the availability of cysteinylglycine produced during GSH catabolism. Furthermore, we showed that cisplatin forms adducts with cysteinylglycine 10 times more rapidly than with GSH, and that these adducts were formed only in the extracellular medium of HeLa GGT cells. This extracellular mechanism could at least partially account for the increased resistance of GGT-rich cells to cisplatin.     

银杏悬浮细胞对酯蟾毒配基3-羟基的选择性异构化反应

目的　利用银杏悬浮细胞对酯蟾毒配基进行结构修饰。方法　利用只含生长素 2 ,4 D的MS培养基诱导银杏嫩叶 ,使细胞脱分化形成愈伤组织 ,然后将愈伤组织转移至含一定浓度 6 BA ,NAA ,和 2 ,4 D的液体MS培养基中以形成悬浮细胞。把酯蟾毒配基加入生长状态良好的悬浮细胞中转化四天。提取出溶解于液体相的转化产物 ,采用硅胶吸附柱层析法 ,以石油醚和丙酮为展开体系进行梯度洗脱 ,然后对转化产物进行分离纯化。结果　经过四天转化 ,得到一个转化产物 ,转化率达 4 0 % ,通过对转化产物的质谱 ,核磁共振氢谱和碳谱等波谱数据进行分析 ,并与有关文献进行对比 ,证明转化产物为 3 表 酯蟾毒配基。结论以银杏悬浮细胞作为一种生物酶体系 ,可以把来源于动物的蟾蜍甾烯类化合物酯蟾毒配基转化成 3 表 酯蟾毒配基。     

Influence of pH on the cytotoxic activity of chlorambucil

The cytotoxic activity of chlorambucil as a function of pH was investigated in P388 tumor cells growing in static suspension culture. A decrease in extracellular pH from 7.8 to 7.2 was associated with a decrease in intracellular pH from 7.92 to 7.55. The cytotoxic potency of chlorambucil increased as the extracellular pH decreased; IC99 values were 20 and 60 microM when the extracellular pH was 7.2 and 7.8 respectively. Covalent binding to cellular macromolecules was about 1.9 times greater at pH 7.2 relative to that at pH 7.8. These results suggest that pH may be an important determinant of the oncotoxic specificity of chlorambucil, and that the cytotoxic activity of this agent could be selectively directed toward tumor cells by the selective manipulation of intracellular and extracellular pH. A potential influence of intracellular and extracellular pH on cytotoxic, mutagenic, carcinogenic, and teratogenic potencies of other chemicals is also suggested. Additionally, these investigations demonstrate the importance of carefully controlling pH throughout the drug exposure period when evaluating the relative potency of potential cytotoxic, mutagenic, carcinogenic, and teratogenic agents in cell or organ culture.     

New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development,standard operation procedures

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.     

Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium.

1. Pure populations of rat peritoneal mast cells were used to study cellular potassium uptake. The radioactive potassium analogue, 86rubidium, was used as a tracer for potassium for measurements of the activity of the cellular potassium uptake process. 2. The ouabain-sensitive and the ouabain-resistant potassium (86rubidium) uptake of mast cells incubated in the presence of calcium, 1 mmol l-1, were very low, 52 and 147 pmol per 10(6) cells min-1. 3. Calcium-deprivation of the cells uncovered a large capacity ouabain-sensitive potassium (86rubidium) uptake mechanism. The activity of the uptake mechanism was decreased by reintroduction of calcium into the cell suspension, and it was dependent on cellular energy metabolism, temperature and pH. 4. The potassium (86rubidium) uptake of mast cells incubated in a calcium-free medium occurs through an active and ouabain-sensitive mechanism that has the nature of an enzyme, and it is mediated by the Na+ -K+ pump located in the plasma membrane. It is demonstrated that the activity of the Na+ -K+ pump mechanism is inhibited by low concentrations of extracellular calcium (0.1-1.2 mmol l-1). The possibility is discussed that calcium-deprivation may increase the pump activity by increasing the permeability of the plasma membrane for Na+.     

Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity.

The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.     

紫球藻胞外多糖的分离及体外抗乙肝病毒活性的初步研究

目的 研究紫球藻胞外多糖的分离及体外抗乙肝病毒活性。方法 紫球藻培养液通过离心、浓缩、透析、醇析、脱蛋白、冷冻干燥等步骤从中分离出胞外多糖；采用ELISA和MTT法测定紫球藻胞外多糖对2215细胞分泌e抗原（HBeAg）的影响。结果 元素分析表明，胞外多糖舍N：0．82％、C：32．91％、H：6．19％；氨基酸分析表明，含有17种氨基酸，含量为2．49％。红外光谱和紫外光谱分析表明，所分离的胞外多糖具有多糖的特征吸收峰，糖环为吡喃环，舍有硫酸酯基团。该胞外多糖对2215细胞分泌的HBeAg有不同程度的抑制作用，其治疗指数（TI）大于2。结论 紫球藻胞外多糖在体外具有抗乙肝病毒作用。     

Development of in vitro techniques for the important medicinal plant Veratrum californicum

VERATRUM CALIFORNICUM (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An IN VITRO culture system for somatic embryogenesis and green plant regeneration of VERATRUM CALIFORNICUM was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The IN VITRO plantlets contained the steroid alkaloids cyclopamine and veratramine. This IN VITRO system will form the basis for metabolic engineering of VERATRUM cells in the context of biotechnological production of pharmaceutically important secondary metabolites. DMSO:dimethyl sulfoxide fw:fresh weight NAA:naphthaleneacetic acid 2,4-D:2,4-dichlorophenoxyacetic acid picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid dicamba:3,6-dichloro-2-methoxybenzoic acid.     

体外胁迫促进青蒿素在黄花蒿培养细胞中合成的研究

在黄花蒿细胞悬浮培养合成青蒿素的过程中添加微生物刺激剂和微生物胸外酶刺激剂,测定刺激剂对青蒿素合成的影响。采用的微生物刺激剂为霉菌ＡＳ３４,３０９和ＡＳ３,３４６、酵母Ｋluyberomyces fragilis及细菌ＡＳ１,３９８的提取物,微生物胸外酶刺激剂为果胶酶、纤维素酶及半纤维素酶。其中酵母Ｋluyberomyces fragilis提取物和果胶酶对青蒿素生物合成具有明显的刺激作用。用酵母Ｋluyberomyces fragilis提取物（２０％）处理黄花蒿培养细胞３天,青蒿素合成量达到每克黄花蒿干细胞３２０μｇ,比未刺激细胞的青蒿素合成量提高３８％;将果胶酶（０.２％）添加到青蒿素合成培养基中处理黄花蒿培养细胞２天以上,青蒿素合成量达到每克黄花蒿干细胞７４０μｇ,比未刺激细胞的青蒿素合成量提高３.０８倍。     

真菌诱导子对胡桐悬浮培养细胞产生红厚壳素的影响

目的探讨真菌诱导子对胡桐悬浮培养细胞产生红厚壳素的影响。方法通过对胡桐叶斑病病菌的分离培养,制成真菌诱导子补加到胡桐细胞悬浮培养基中,考察不同浓度、不同加入时间对胡桐细胞生物量及红厚壳素产量的影响。结果S-I菌株诱导胡桐CR2细胞合成红厚壳素的最佳浓度为60 mg GE·L^-1,诱导子在细胞生长静止期初期(即培养d 18)加入对红厚壳素产量影响最大,可使产量提高27%,并促进了红厚壳素向胞外分泌。结论 壳多孢菌的添加能有效提高胡桐细胞悬浮培养体系中红厚壳素的产量。     

Production of eurycomanone from cell suspension culture of Eurycoma longifolia

Context: Eurycomanone is found in the Eurycoma longifolia Jack (Simaroubaceae) tree, exhibits significant antimalarial activity, improves spermatogenesis, suppresses expression of lung cancer cell tumour markers and regulates signalling pathways involved in proliferation, cell death and inflammation.

Objectives: Establishment of cell suspension culture of E. longifolia to determine the eurycomanone accumulation during cultures.

Materials and methods: Callus of E. longifolia was cultured in MS medium supplemented with 0.8% agar, 30/L sucrose, 1.25?mg/L NAA and 1?mg/L KIN for biomass production. Cell suspension culture was established by transferring friable calli to the same medium without agar. Eurycomanone content during cell culture was determined by HPLC with a C18 column, flow rate of 0.8?mL/min, run time of 17.5?min, detector wavelength of 254?nm. The stationary phase was silica gel and the mobile phase was acetonitric:H₂O. Roots of 5 year-old trees were used as the control.

Results: The cells from 3?g of inoculum increased in biomass with a maximum value of 16?g fresh weight (0.7?g dry weight) at 14th day of culture. The cell growth then decreased from day 14 to day 20. Eurycomanone was produced during culture from the beginning to 20th day, its highest content (1.7?mg/g dry weight) also obtained at 14th day (the control is 2.1?mg/g dry weight).

Discussion and conclusions: Cell suspension culture of E. longifolia is a suitable procedure to produce eurycomanone. The yield of eurycomanone biosynthesis in 14 days-old cells are relatively high, approximately 0.8 times the control.