The use of 5-fluorocytosine and ketoconazole in the culture of the erythrocytic stages ofPlasmodium falciparum and some tumor cell lines

Summary In vitro culture systems are often contaminated by bacteria and fungi. It is therefore often necessary to supplement culture media with agents such as penicillin/streptomycin, gentamycin or amphotericin B. The latter cannot be used in the in vitro culture of erythrocytic stages ofP. falciparum, and thus anti-fungal agents have not been regularly used in this system. We describe the prophylactic use of 5-fluorocytosine (5-FC) and ketoconazole (KTZ) in tissue cultures at concentrations up to 300 and 10 g/ml respectively which have no effect on the growth ofP. falciparum (FCR-3 strain). A melanoma cell line (C32) and a line of uterine carcinoma (C41) were also unaffected by similar concentrations of 5-FC and KTZ. When dissolved in complete culture medium (RPMI 1640) with 10% human plasma, the minimum inhibitory concentration of 5-FC for a susceptible strain ofCandida remained below 2 g/ml. These experiments suggest that 5-FC (at 50 g/ml) alone or in combination with KTZ (at 1 g/ml) is a useful addition to the armamentarium of antimicrobials available to the tissue culture biologist for a variety of cell culture systems.     

Isometachromin, a new cytotoxic sesquiterpenoid from a deep water sponge of the family Spongiidae.

Isometachromin (1), a new sesquiterpene-quinone that is related structurally to metachromin C (2), and the known compounds ilimaquinone (3) and 5-epi-ilimaquinone (4), were isolated from a deep water sponge in the family Spongiidae; the structure of isometachromin was elucidated by spectral methods. Isometachromin exhibits in vitro cytotoxicity against the human lung cancer cell line A549 (IC50 = 2.6 micrograms/ml), but not against P388 murine leukemia (IC 50 > or equal to 10 micrograms/ml) and also exhibits antimicrobial activity.     

Influence of beta-aminoproprionitrile (BAPN) on cell growth and elastic fiber formation in cultures of auricular chondrocytes

Auricular chondrocytes isolated from 4-day-old rabbits and grown in vitro for 14 days, proliferated rapidly and produced a conspicuous network of elastic fibers. Beta-aminoproprionitrile (BAPN), which in vivo inhibits cross-linking of elastin, decreased the formation of elastic fibers at a concentration of 10-20 micrograms/ml and prevented formation at 40 micrograms/ml. At a concentration of 5 micrograms/ml only the so-called patches of elastin appeared to be absent. The inhibitory effect of BAPN on cell growth did not exceed 10%, which indicates that BAPN is only slightly harmful to auricular chondrocytes and can safely be used in studies on elastin deposition by these cells in vitro.     

In vitro malignant transformation of fetal hamster brain cells by methylnitrosourea]

Using primary cultures originating from 14 day old fetal Hamster brain, we have obtained a cell line with glial morphology. These cell remain non transplantable during the first year of in vitro culture, but undergo spontaneous transformation during the second year. Following prolonged contact of the glial-like cells with 25 to 50 microgram/ml of methylnitrosourea (MNU), a malignant transformation is observed 5 months after the treatment. The lowest concentrations of (MNU) do not cause malignant transformation, but seem to inhibit (or postpone) the spontaneous transformation. After MNU treatment, cells retain their glial nature.     

Elevated glucose levels influence in vitro hatching,attachment, trophoblast outgrowth and differentiation of the mouse blastocyst

Summary A glucose concentration of 3.5 mg/ml is optimal for in vitro embryo attachment and trophoblastic cell outgrowth. Raising the concentration above 3.5 mg/ml does not improve embryo culture and can at certain concentrations be detrimental to embryo development.     

Effect of actinomycin D on Trypanosoma cruzi

Viable metacyclic forms of T. cruzi, Y strain, treated with an adequate dose of actinomycin D (50 micrograms Act-D/ml/10(7) parasites/ml for 72 h at 28 degrees C) showed the following properties: 1) they lost their ability to replicate in culture medium, in blood and in tissues of normal mice and were no longer able to incorporate tritiated thymidine; 2) they could not penetrate into Vero cells and could not replicate inside normal macrophages; 3) they retained their immunogenicity and the ability to protect mice against a virulent infection; 4) they did not induce histological lesions as described in chronic experimental Chagas' disease.     

刚地弓形虫培养上清抑制THP-1细胞增殖及诱导凋亡的研究

摘要：目的提取弓形虫体外细胞共培养上清,并研究上清对人急性单核细胞白血病细胞THP-1增殖及凋亡的影响。方法收集对数生长期的THP-1细胞以5X10^7／ml细胞浓度接种于不同培养瓶中,对照组加入含10％胎牛血清的RPMll640,实验组加入相同体积不同数量（2×10^7／ml、4X10^7／ml、8×10^7／m1）弓形虫速殖子培养上清,采用四甲基氮噻唑蓝（MTY）法检测吸光度（A490值）并计算THP-1细胞增殖抑制率;倒置显微镜下观察细胞形态变化;Annexin-V-FITC／PI染色细胞后上流式细胞仪检测各个时间点细胞凋亡率变化,以Western印迹方法分析凋亡相关蛋白Bax、Bcl-2的表达或活性。结果MTY法检测结果弓形虫培养上清呈时间剂量依赖性抑制THP-1细胞株增殖,倒置显微镜下观察处理组细胞有发泡现象和凋亡小体出现。流式细胞仪检测弓形虫感染后的THP-1细胞凋亡率较对照组有升高趋势（P〈0．05）,呈量效依赖性,Westernblot检测刚地弓形虫培养上清作用于THP-l细胞48h后实验组的Bax、Bcl-2蛋白表达较对照组的比值分别有明显的升高与降低（P〈0．05）。结论刚地弓形虫速殖子培养上清对体外培养THP-l细胞增殖有明显的抑制作用,并可诱导THP-1细胞凋亡。     

Phenytoin-induced DNA synthesis and inositol 1,4,5-trisphosphate formation in L-929 fibroblasts

Culture of L-929 fibroblasts in the presence of phenytoin (2.5-5.0 micrograms/ml) increased DNA synthesis, as indicated by increased [3H]thymidine uptake, while a higher dose (20 micrograms/ml) inhibited DNA synthesis. In like manner, a low dose of phenytoin (5.0 micrograms/ml) was effective in increasing inositol 1,4,5-trisphosphate formation while a higher dose (10 micrograms/ml) tended to inhibit this activity. These data suggest that the formation of inositol phosphate second messengers may play a role in phenytoin-induced fibroblast proliferation and connective tissue growth.     

Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes

A Ginkgo biloba extract (Gbe) containing flavonoids, among other compounds, was tested for the release of activated oxygen species (O-2, H2O2, OH.) during the stimulation of human neutrophils (PMNs) by a soluble agonist. The extract slows down O2 consumption (respiratory burst) of stimulated cells by its inhibitory action on NADPH-oxidase, the enzyme responsible for the reduction of O2 to O-2. Consequently, superoxide anion (O-.2) and hydrogen peroxide (H2O2) production is significantly decreased when the PMNs stimulation is done in the presence of the extract at concentrations of 500, 250 and 125 micrograms/ml. Moreover, the hydroxyl radical generation (OH.) is very much decreased at concentrations as low as 15.6 micrograms Gbe/ml, which indicates that the extract also has free radical scavenging activity. Gbe is able at least to reduce very severely the activity of myeloperoxidase contained in neutrophils. This enzyme, secreted into the intra and extracellular medium, catalyzes the oxidation of chloride (Cl-) by H2O2 to yield strong oxidants (HOCl, chloramines) which are implicated in inflammatory processes.     

Exogenous fibronectin modifies the aggregation of collagen-stimulated human platelets

Fibronectins (FN) are adhesive glycoproteins whose role in platelet aggregation is unclear. Addition of 3, 6 and 12 micrograms/ml of human plasma FN in vitro to isolated human platelets, which had been freed from plasma FN by gel filtration and subsequently stimulated with collagen, inhibited the last stage of platelet aggregation. With 3 and 6 micrograms/ml of FN a shortening of the lag-time was also observed. These data showed that FN may play a role in platelet-collagen interaction as well as in platelet-platelet interaction.     

Persistence of added retinoids in organ culture media during induction of mucous metaplasia and glandular morphogenesis in hamster cheek pouches

The retinoid concentration (determined colorimetrically) did not change significantly in retinyl acetate-supplemented (6 micrograms/ml) Eagle's Minimal Essential Medium containing 10% fetal calf serum when stored at -20 or 4 degrees C over 7 days. After the medium was incubated at 37 degrees C for 48 h, 37-49% of the retinoid remained, whether or not tissue (neonatal Syrian hamster cheek pouch) was present, and irrespective of explant age. The normal retinoid level in the tissue was approximately 0.25 micrograms per gram. Therefore, neonatal hamster cheek pouches, incubated in medium with the addition of 6 micrograms of retinyl acetate per ml of medium and undergoing mucous metaplasia and some mucous gland morphogenesis, were continually being exposed to retinoid levels which, though gradually decreasing, remained well above their normal physiological level.     

Antifibrinolytic properties of oxyphenbutazone in vitro

Summary The authors found that oxyphenbutazone (Tanderil^®) added to culture medium, in amounts giving a final concentration of 10–100 g/ml, causes a decrease in the liberation of fibrinolytic agents from explants of various tissues cultured in vitro.     

氧化苦参碱对人宫颈癌SiHa细胞株增殖及凋亡的影响

目的研究氧化苦参碱对体外人宫颈癌SiHa细胞株增殖活性及凋亡的影响。方法对人宫颈癌SiHa细胞株进行体外培养,经氧化苦参碱处理的细胞组为实验组,未经处理组为对照组。采用MTT细胞存活实验检测氧化苦参碱对入宫颈鳞癌SiHa细胞的增殖影响,并计算IC50;倒置相差显微镜下观察不同浓度的氧化苦参碱作用48h后SiHa细胞的形态改变;Hoechst33258染色法和Western blot法检测氧化苦参碱对SiHa细胞核及凋亡相关蛋白(P53、Bax及Bcl-2)表达水平的影响。结果 MTT结果显示氧化苦参碱剂量-时间依耐性抑制人宫颈癌SiHa细胞的体外增殖(P0.05),计算24 h、48 h和72 h的IC50分别为(1 028.41±3.57)μg/ml、(701.72±6.01)μg/ml和(406.88±2.15)μg/ml;氧化苦参碱处理48 h后,SiHa细胞的形态特征及数目发生显著变化,且随氧化苦参碱浓度的增加而愈加明显;Hoechst33258染色证实氧化苦参碱处理后SiHa细胞发生凋亡,可见典型的凋亡特征;Western blot结果证实氧化苦参碱(700μg/ml、1 600μg/ml)处理48 h后,和对照组比较,药物处理组细胞凋亡周期蛋白P53、Bax表达上调(P0.05),而Bcl-2表达下调(P0.05),Bax/Bcl-2的比率明显增加(P0.05)。结论氧化苦参碱可抑制体外人宫颈癌SiHa细胞的体外增殖活性发挥其抗肿瘤作用,其作用机制可能诱导SiHa细胞的凋亡有关。     

Failure of somatostatin to influence experimental tumor cell growth in vivo and in vitro

The influence of somatostatin on tumor cell growth was studied in vivo in mice (sarcoma 180 ascites tumor and Lewis lung tumor) and in vitro on nontransformed and polyoma-transformed cell lines. 4 or 20 micrograms/100 g of cyclic somatostatin and 4 micrograms/100 g of linear protamin Zn-bound somatostatin were injected s.c. twice daily in the in vivo study. Cyclic somatostatin (1, 4 or 10 micrograms/ml) was added twice daily to the cell cultures. Somatostatin administration influenced neither the survival of animals nor the growth rate of cultured cell lines.     

Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells

Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized.     

运用磁珠分选法建立免疫细胞共培养体系

目的以相互作用的几种免疫细胞上共同表达的抗原物质为标志,运用磁珠分选技术,从慢性乙型肝炎患者外周血单个核细胞中同时获得并建立体外多细胞共培养体系,体外观察细胞之间的相互作用.方法采用梯度离心法分离30例慢性乙肝病毒感染者外周血单个核细胞(PBMC),采用免疫磁珠分选法分离获得 CXCR5+细胞,通过流式细胞仪检测 CXCR5+细胞纯度和组成,并将 CXCR5+细胞进行体外培养.在分离得到的 CXCR5+细胞的基础上,将 CXCR5+细胞分为3组,分别为对照组:不加任何刺激;实验组一:加入 IL 21刺激培养,实验组二:与人肝癌细胞系(HepG2)2215细胞混合培养.体外培养一周后,流式细胞仪检测实验组与对照组细胞 B细胞数量变化,ELISA测定培养体系中上清液 HBe抗体的含量.结果1)用流式细胞仪鉴定 CXCR5+细胞的纯度和构成:CXCR5+细胞纯度为85.5±5.8%,其中 Tfh细胞占23.8±7.4%,B细胞占35.6±7.6%;2)培养7天后,IL 21刺激组 B细胞百分率为47.2±1.8%,与(HepG2)2215混合培养组 B细胞百分率为40.2±3.5%,空白对照组 B细胞百分率为36.6±7.5%,IL 21刺激组与对照组,(HepG2)2215混合培养组与对照组 B细胞百分率均有显著差异(p<0.05);3)ELISA检测培养液上清 HBeAb定量,IL 21刺激组为0.668±0.094pg/ml,空白对照组为0.378±0.088pg/ml,两组有显著差异(p<0.05).结论使用间接免疫磁珠分离法可成功建立 CXCR5+B淋巴细胞和 Tfh细胞的共培养体系,为进一步体外研究细胞之间相互关系奠定基础     

The effect of nicotine on ethanol-induced gastric ulcers in rats

Nicotine, in concentrations of 5 and 25 micrograms/ml drinking water, given ad libitum for 10 days, dose-dependently increased lesion formation and worsened ethanol-induced ulceration in rat stomachs. Daily fluid intake and b.wt gain were not adversely affected by nicotine pretreatment.     

Heat evolution of cultured human keratinocytes

Summary The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm ^tm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83±12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134±35) pW/cell was obtained when the transformed kerationocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.     

Postimplantation embryo culture for the assessment of the teratogenic potential and potency of compounds

Whole rat embryos cultured during the early stages of organogenesis were subjected to a panel of selected chemicals. Of seventeen known in vivo teratogens, seventeen also induced specific malformations in embryos grown in culture. Of ten chemicals which were reported to be negative in in vivo rat teratogenicity studies, eight also did not provoke dysmorphogenic effects in vitro. Of five additionally tested retinoids, all induced multiple malformations. However, concentrations used to induce these effects varied considerably, isotretinoin inducing malformations at 10(-5) M and arotinoid at 10(-11) M. The results indicate qualitatively as well as quantitatively a high predictability of this in vitro system and suggest that the postimplantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.     

Presence of oncornavirus-like particles in the P388 murine leukemic cell line

A culture of P388 murine lymphoblastoid cells has been shown to contain type C oncornavirus-like particles budding at the plasma membrane. Occasionally intracytoplasmic type A and immature type B particles were also observed by electron microscope techniques. The discovery of oncornavirus-like particles in the P388 cell line increases the utility of this neoplastic system for detecting potential antineoplastic agents.