IAPs Regulate Distinct Innate Immune Pathways to Co-ordinate the Response to Bacterial Peptidoglycans

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小鼠胚胎与子宫单层上皮细胞共培养的研究

本文报道建立了小鼠胚胎与小鼠子宫单层上皮细胞体外共培养系统。结果揭示;小鼠胚胎与 子宫单层上皮细胞共培养可以促进胚胎的发育、粘附和扩展;如果培养液中加入 ３、６７ × １０－６ｍｏｌ／Ｌ １７β－雌二醇,可以显著提高胚胎在共培养系统中的发育率、粘附率和扩展率。以上结果表明：小鼠 胚胎与小鼠子宫单层上皮细胞共培养系统是研究胚泡着床机理较理想的研究手段。     

EGF-Induced sustained tyrosine phosphorylation and decreased rate of down-regulation of EGF receptor in PC12h-R cells which show neuronal differentiation in response to EGF

PC12h-R cell, a subclone of PC12 cells, exhibited a neuron-like phenotype, including neurite outgrowth and increased acetylcholinesterase activity, in response to epidermal growth factor (EGF) as well as nerve growth factor (NGF). We examined the mechanism by which EGF induced the neuronal differentiation in PC12h-R cells. The EGF-induced neuronal differentiation of PC12h-R cells was not blocked by K252a, whereas that induced by NGF was. EGF induced sustained tyrosine phosphorylation of the EGF receptor in PC12h-R cells, but not in the parent PC12h cells, which do not show neuronal differentiation in response to EGF. In addition, the rate of EGF-induced down-regulation of the EGF receptor in PC12h-R cells was decreased compared with that in PC12h cells. Furthermore, we found that the duration of EGF-induced tyrosine phosphorylation of the EGF receptor in PC12h-R cells was similar to that of NGF-induced tyrosine phosphorylation of p140 ^trkA in PC12h cells. The EGF-induced phosphorylation of the EGF receptor in PC12h cells was less sustained than that of p140 ^trkA by NGF in PC12h cells. These findings suggested that the EGF-induced neuronal differentiation of PC12h-R cells is due to the sustained activation of the EGF receptor, resulting from the decreased down-regulation of the EGF receptor and that the duration of the receptor tyrosine kinase activity determines the cellular responses of PC12 cells. We concluded that sustained activation of the receptor tyrosine kinase induces neuronal differentiation, although transient activation promotes proliferation of PC12 cells. Special issue dedicated to Dr. Hans Thoenen.     

Effect of interferon γ and tumour necrosis factor α on sensitivity to cisplatin in ovarian carcinoma cell lines

This study aimed to investigate whether the biological response modifiers (BRM) interferon (IFN) and tumour necrosis factor (TNF) could enhance the cytotoxic action of cisplatin on ovarian tumour cells in vitro. The sensitivity of four cell lines (OAW42, GG, JAM and PE01) to drugs and drug combinations was tested by a radiolabelled-thymidine incorporation assay. Cell lines demonstrated a range of sensitivity to cisplatin and the innate cytotoxic effect of each of the BRM. When IFN was used in combination with cisplatin, a significant enhancement of cisplatin toxicity occurred in three of four cell lines. TNF demonstrated such an effect in two cell lines but diminished the toxicity of cisplatin in one cell line. A purely additive effect of the agents may explain the enhanced toxicity of cisplatin in some of these cases. However, in one cell line at least (PEO1), both TNF and IFN demonstrated a clearly synergistic effect with cisplatin. These BRM used in conjunction with cisplatin may provide better antitumour regimen than cisplatin alone in some patients with ovarian cancer, but the response is likely to be heterogeneous between patients.     

Active cell membrane mechanisms involved in the exclusion of RH 123 allow distinction between normal and tumoral cells

Human cell lines derived from three epithelial carcinomas (CaSki, HeLa, SiHa), one B lymphoma (BL60), one promyelocytic (HL60), one monocytic (U937) leukemia, one chronic myelogenous leukemia (sensitive K562S; multichemoresistant K562R) and normal human skin fibroblasts were compared for their capacity of staining with rhodamine 123 (Rh 123) and their kinetics of dye exclusion. Cells were exposed for 30 min to 10 g/ml of Rh 123 in culture medium; fluorescence intensity was measured by flow cytometry immediately or 1, 2, 3 and 4 h after staining. The highest fluorescence intensity was observed in carcinoma cell lines; there was no incorporation in multichemoresistant K562R cells. Exclusion of Rh 123 was evaluated from 0 to 4 h, both by flow cytometry and by fluorimetry. Fluorescence intensity measured by flow cytometry decreased slightly in carcinoma and leukemia cells and rapidly in fibroblasts. In all cell lines Rh 123 exclusion was inhibited by 40 mol/L verapamil and 5 mmol/L probenecid. Thus, incorporation and exclusion of Rh 123 allows distinction between normal and tumoral cells; moreover, inhibition of exclusion by verapamil and probenecid favors the involvement of active cell membrane mechanisms in the exclusion process.Abbreviations PBS phosphate-buffered saline - Rh 123 rhodamine 123     

Engineering challenges in high density cell culture systems

High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO₂, a significant accumulation of dissolved CO₂ is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Biotechnological Production of Plant-Based Insecticides

ABSTRACT:?

The demand for natural and nonpersistent insecticides is increasing day by day. Plant cell cultures could be an alternative to conventional methods of production of insecticides from field-grown plants. In vitro cultured plant cells produce a wide array of insecticides as a part of their secondary metabolism. Their ability to synthesize key enzymes and the manipulation of these could lead to the enhanced production of many insecticides of industrial importance. The development of a high-yielding hairy root culture system for thiophenes, nicotine, and phytoecdysones is of considerable interest. In this article, the current literature on various factors that influence the growth, production, and secretion of six insecticidal compounds, namely, pyrethrins, azadirachtin, thiophenes, nicotine, rotenoids, and phytoecdysones which have been prospects for the scale-up of cell cultures, genetic engineering to obtain transgenic plants, and metabolically engineered plants for increased production of bio-molecules, has been discussed. Environmental safety clearance and the future prospects of application of bio-molecules for plant-derived insecticides are presented.