In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

Significant Differences in the Development of Acquired Resistance to the MDM2 Inhibitor SAR405838 between In Vitro and In Vivo Drug Treatment

SAR405838 is a potent and specific MDM2 inhibitor currently being evaluated in Phase I clinical trials for the treatment of human cancer. Using the SJSA-1 osteosarcoma cell line which harbors an amplified MDM2 gene and wild-type p53, we have investigated the acquired resistance mechanisms both in vitro and in vivo to SAR405838. Treatment of SJSA-1 cells with SAR405838 in vitro leads to dose-dependent cell growth inhibition, cell cycle arrest and robust apoptosis. However, prolonged treatment of SJSA-1 cells in vitro with SAR405838 results in profound acquired resistance to the drug. Analysis of in vitro-derived resistant cell lines showed that p53 is mutated in the DNA binding domain and can no longer be activated by SAR405838. Treatment of the parental SJSA-1 xenograft tumors with SAR405838 in mice yields rapid tumor regression but the tumors eventually regrow. Culturing the regrown tumors established a number of sublines, which showed only modest (3–5 times) loss of sensitivity to SAR405838 in vitro. Sequencing of the p53 showed that it retains its wild-type status in these in vivo sublines, with the exception of one subline, which harbors a single heterozygous C176F p53 mutation. Using xenograft models of two in vivo derived sublines, which has either wild-type p53 or p53 containing a single heterozygous C176F mutation, we showed that while SAR405838 effectively achieves partial tumor regression in these models, it no longer induces complete tumor regression and tumors resume growth once the treatment is stopped. Harvesting and culturing tumors obtained from a prolonged treatment with SAR405838 in mice established additional in vivo sublines, which all contain a single heterozygous C176F mutation with no additional p53 mutation detected. Interestingly, SAR405838 can still effectively activate p53 in all sublines containing a single heterozygous C176F mutation, with a moderately reduced potency as compared to that in the parental cell line. Consistently, SAR405838 is 3–5 times less effective in all the in vivo derived sublines containing a single heterozygous C176F p53 mutation than in the SJSA-1 parental cell line in assays of cell growth and apoptosis. Computational modeling suggested that a p53 tetramer containing two wild-type p53 molecules and two C176F mutated molecules can maintain the structural stability and interactions with DNA by formation of additional hydrophobic and cation-π interactions which compensate for the loss of sulphur-zinc coordination. Our data thus show that SJSA-1 tumor cells acquire very different levels of resistance in vitro and in vivo to the MDM2 inhibitor SAR405838. Our present study may have a significant implication for the investigation of resistant mechanisms for other classes of anticancer drugs.     

In Vitro Induced Anaerobic Resistance to Metronidazole In Trichomonas Vaginalis

ABSTRACT. Resitance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 μ/ml) for 12 to 21 months. the resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 μ/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 μ/ml at 48-h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 μ/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 μ/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 μ/ml metronidazole. the strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [¹⁴C]-metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.     

In Vitro Selection for Improved Plant Resistance to Toxin-Producing Pathogens

Simultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin‐producing pathogens, mode of phytotoxic action, and host‐ and non‐host‐selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.     

Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies most higher-order sphingolipids. Surprisingly, recent studies have shown that certain chemotherapeutics and other drugs inhibit Des1, giving rise to a number of sphingolipids that lack the characteristic double bond. In order to assess the effect of an altered sphingolipid profile (via Des1 inhibition) on cell function, we generated isogenic mouse embryonic fibroblasts lacking both Des1 alleles. Lipidomic profiling revealed that these cells contained higher levels of dihydroceramide than wild-type fibroblasts and that complex sphingolipids were comprised predominantly of the saturated backbone (e.g. sphinganine vs. sphingosine, dihydrosphingomyelin vs. sphingomyelin, etc.). Des1 ablation activated pro-survival and anabolic signaling intermediates (e.g. Akt/PKB, mTOR, MAPK, etc.) and provided protection from apoptosis caused by etoposide, a chemotherapeutic that induces sphingolipid synthesis by upregulating several sphingolipid biosynthesizing enzymes. These data reveal that the double bond present in most sphingolipids has a profound impact on cell survival pathways, and that the manipulation of Des1 could have important effects on apoptosis.     

Embryological Studies on In Vitro Pollination in Rice

n vitro pollination and its embryological studies were carried out in two japonica cultivars of rice (Oryza sativa L.), “Chunjiang 05" and “95046". N6 basic medium supplemented with different exogenous hormones was used for ovary culture after in vitro pollination. The main results were as follows: (1) both cultivars were induced to set kernels after in vitro pollination. The frequency of seedset was 52.1%, including 28.4%normal embryo development, 2.2% abnormal embryo development and 21.5% callus formation. (2) The processes of embryo and endosperm development after in vitro pollination were basically as normal as those in vivo , except there was some retardation in the first division of zygotes and primary endosperm nuclei as well as in their subsequent development. However, both kernels and plantlets could be produced finally. (3) A few abnormal embryos were observed, for instance, proembryos with elongated suspensor and vacuolated proembryos. (4) Two types of calli in the cultured ovaries appeared, namely, the compact callus and the fragile callus, which were able to differentiate into adventitious buds and roots.     

水稻离体授粉的胚胎学研究

采用2个水稻（Oryza sativa L.)品种“春江05”（早粳）和“95046”（晚粳）,对离体授粉过程中的胚胎学进行了详细的研究。结果表明：（1）2个品种均胡离体授粉结实,平均结实率为52．1％,其中28．4％胚胎发育正常,2．2％胚胎发育异常,21．5％愈伤组织化;（2）离体授粉时的胚胎发育途径和体内自然发育基本相同,中是合子和初生胚乳核首次启动分裂及以后的发育均较延缓,但最终也能萌发成幼苗。（3）观察到具细长胚柄的原胚及液泡化原胚等异常的胚胎类型;（4）子房内形成的愈伤组织分为致密和松散两种类型,二均可化出不定芽和不定根,还对离体授粉的方法,离体授粉中正常和异常的胚胎发育途径进行了讨论。     

In Vitro Studies with 5-Fluorocytosine

5-Fluorocytosine, an antifungal agent with potential value as a chemotherapeutic agent, is being evaluated in the treatment of human cryptococcosis. In vitro studies with this agent have been hindered by the fact that it is inhibited significantly in the presence of partially degraded biological substances. This loss of activity is presumed to result from a competitive inhibition between the agent and its natural analogues. Procedures are described for in vitro studies with 5-fluorocytosine. These include methods for susceptibility testing and a bioassay for 5-fluorocytosine in biological fluids. Minimal inhibitory and minimal fungicidal concentrations of 5-fluorocytosine for Cryptococcus neoformans were usually in the range of 0.46 to 3.9 mug/ml and 3.9 to 15.6 mug/ml, respectively. Corresponding values for Candida albicans were 0.46 to 3.9 mug/ml and 15.6 mug/ml or greater, respectively. Strains of C. neoformans and C. albicans resistant to greater than 1,000 mug/ml were encountered both after exposure to the drug and in the absence of any known exposure. Bioassays of specimens from patients treated with 5-fluorocytosine indicated that serum and cerebrospinal fluid concentrations of 10 to 30 mug/ml and 8 to 20 mug/ml, respectively were readily achieved with a dosage of 100 mg per kg per day.     

昆虫对Bt毒素产生抗性的分子机制

简述了近年来国内外对昆虫产生抗性的模式及其分子机制的研究进展,对与昆虫抗性有关的中肠蛋白酶和多种受体进行了较深入的讨论。展望了持续有效地利用Bt生物制剂和保持转Bt基因作物的抗虫性的前景。     

In Vitro Studies on the Initial Reactions of Anaerobic Ethylbenzene Mineralization

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) [mg of protein](-1) and an apparent K(m) for ethylbenzene of approximately 60 microM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.     

In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

In Vitro Studies on the Isolation of Genital Mycoplasmas from Blood

A method is described for the isolation of genital mycoplasmas from clinical blood cultures.