Modulation of DNA precursor pools, DNA synthesis, and ultraviolet sensitivity of a repair-dificient CHO cell line by deoxycytidine

The presence of 2 mM deoxycytidine (CdR) in growth medium substantially increased the deoxycytidine triphosphate (dCTP) and deoxuthymidine triphosphate (dTTP) pools in a Chinese hamster ovary cell line, CHO-K1, and in a radiation-sensitive mutant, xrs-5, derived from it (Jeggo et al., 1982). We observed significant differences in alkaline-sucrose gradient profiles of pulse-labeled DNA from unirradiated CHO-K1 and xrs-5 cells. For the latter cell line, a sizable fraction of the DNA synthesized during 5 or 10 min of growth subsequent to a 5-min radiolabeling period was found to co-sediment with large-chromosome DNA. This characteristics of xrs-5 was dramatically reduced by the presence of 2 mM CdR in the culture medium, and the UV resistance of the mutant increased to nearly that of the parent cell line under these culture conditions. These results show that the locus conferring UV-radiation sensitivity to xrs-5 affects DNA replication and that replicative activity and UV-radiation sensivity are jointly modulated by CdR, possibly through its impact on the size of deoxynucleoside triphosphate pools.     

The relationship between precursor and mature forms of the 23 S ribosomal RNA

Summary Oligonucleotide fingerprinting shows the precursor form of the 23S ribosomal RNA fromBacillus megaterium to be larger than its mature counterpart, by some 8 percent, or approximately 250 nucleotides. It can further be shown that the 23SrRNA precursor doesnot contain the 5SrRNA sequence, as had been previously suggested.     

Isoenzyme studies of whole muscle grafts and movement of muscle precursor cells

Summary Isoenzymes of glucose-6-phosphate isomerase (GPI: E.C. 5.3.1.9) were used as markers to determine the origin of cells which give rise to new muscle formed in allografts of whole intact muscle. GPI isoenzymes were also employed to see whether host precursor cells, which have been shown to contribute to muscle formation in grafts of minced muscle, can be derived from muscle lying adjacent to grafts.Excellent muscle regeneration was found in allografts of extensor digitorum longus (EDL) muscle examined after 58 days: 12 of 16 grafts contained 80% or more new muscle. Isoenzyme analysis showed that most, and in 2 instances all, new muscle was derived from implanted donor cells; however, there was strong evidence that in 5 grafts some, or all, new muscle must have resulted from host cells moving into the graft. Although hybrid isoenzyme was not detected this was attributed to factors associated with host tolerance which appear to interfere with fusion between host and donor myoblasts.Isografts of minced muscle were placed next to whole EDL muscle allografts to see if cells from allografts moved into adjacent regenerating tissue. Unfortunately, muscle regeneration in minced isografts was poor; only 3 contained 50% or more new muscle and most contained large amounts of fibrous connective tissue. Only a single isoenzyme band was detected in 11 isografts, but in five instances, the presence of a second band showed that cells from EDL allografts were also present. As no hybrid isoenzyme was detected, it is not known whether these cells which had moved into the regenerating minced grafts were muscle precursors, fibroblasts or some other cell types.     

Ezrin/Radixin/Moesin Are Required for the Purinergic P2X7 Receptor (P2X7R)-dependent Processing of the Amyloid Precursor Protein

The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.     

Enhanced production of the pharmaceutically important polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy

Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA₃ 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four‐week cultures (variant A) were selected to perform the precursor feeding experiment. The L‐phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3‐fold (e.g. p‐coumaric acid – 17.3 fold; casticin – 4.8‐fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16‐fold), p‐coumaric acid (5.3‐fold), rutin (2.8‐fold), caffeic acid (1.5‐fold) and cinaroside (1.5‐fold) than the leaves of its parent greenhouse‐cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p‐coumaric acids.     

南方红豆杉细胞双液相培养中强化紫杉醇生产的研究

研究了南主红豆杉(Taxuschinensisvar.maireiChengetFu)细胞双液相培养中前体饲喂和混合糖源作为生产培养基糖源对细胞生长和紫杉醇合成的影响。结果表明,前体饲喂(苯丙氨酸、苯甲酰胺和醋酸钠)或混合糖(麦芽糖和蔗糖)作为生产培养基糖源对南方红豆杉细胞单液相培养、更重要的是对其双液相培养的紫杉醇产量提高有显著促进作用,而且前体饲喂和混合糖源的协同作用对其双液相培养的紫杉醇产量提高有进一步的促进作用。与对照组(蔗糖作为糖源和无前体的单液相培养)相比,在南方红豆杉细胞双液相培养中,第10天加入前体,第11天加入混合糖源,紫杉醇产量提高4倍多     

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class Ia gene H-2K^b under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2K^b heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-K^b construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models. Mol. Reprod. Dev. 50:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry

The applicability of LC–MS/MS in precursor ion scan mode for the detection of urinary stanozolol metabolites has been studied. The product ion at m/z 81 has been selected as specific for stanozolol metabolites without a modification in A- or N-rings and the product ions at m/z 97 and 145 for the metabolites hydroxylated in the N-ring and 4-hydroxy-stanozolol metabolites, respectively. Under these conditions, the parent drug and up to 15 metabolites were found in a positive doping test sample. The study of a sample from a chimeric uPA-SCID mouse collected after the administration of stanozolol revealed the presence of 4 additional metabolites. The information obtained from the product ion spectra was used to develop a SRM method for the detection of 19 compounds. This SRM method was applied to several doping positive samples. All the metabolites were detected in both the uPA-SCID mouse sample and positive human samples and were not detected in none of the blank samples tested; confirming the metabolic nature of all the detected compounds. In addition, the application of the SRM method to a single human excretion study revealed that one of the metabolites (4ξ,16ξ-dihydroxy-stanozolol) could be detected in negative ionization mode for a longer period than those commonly used in the screening for stanozolol misuse (3′-hydroxy-stanozolol, 16β-hydroxy-stanozolol and 4β-hydroxy-stanozolol) in doping analysis. The application of the developed approach to several positive doping samples confirmed the usefulness of this metabolite for the screening of stanozolol misuse. Finally, a tentative structure for each detected metabolite has been proposed based on the product ion spectra measured with accurate masses using UPLC–QTOF MS.