Glycosylation engineering in Chinese hamster ovary cells using tricistronic vectors

Expression of recombinant glycoproteins carrying the sialyl-Lewis^X epitope requires host cells equipped with appropriate glycosyltransferases. Chinese hamster ovary cells transfected with tricistronic vectors and encoding the resistance marker gene, neoR, in the third cistron and core 2 N-acetylglucosaminyltransferase or 1,3-fucosyltransferase III or IV in either the first or second cistron, respectively, produced both enzyme activities in a constant ratio. A representative clone was transfected with PSGL-1 (P-selectin glycoprotien ligand 1) and conferred P-selectin-binding activity to PSGL-1.     

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y₂ receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y₂ receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y₂ receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y₂ receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y₂ receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y₂ receptors. While ATP and UTP activated the murine and human P2Y₂ receptors with similar potencies (EC₅₀ values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC₅₀ = 9.1-21.2 M) than UTP (IC₅₀ = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.     

The use of internal nitrogen stores in the rhizomatous grass Calamagrostis epigejos during regrowth after defoliation

BACKGROUND AND AIMS: The regrowth dynamics after defoliation of the invasive grass Calamagrostis epigejos were studied. As nitrogen (N) reserves have been shown to play an important role during plant regrowth, the identity, location and relative importance for regrowth of N stores were determined in this rhizomatous grass. METHODS: Plant growth, nitrate uptake and root respiration were followed during recovery from defoliation. Water soluble carbohydrates, nitrate, free amino acids and soluble proteins were analysed in the remaining organs. KEY RESULTS: Nitrate uptake and root respiration were severely reduced during the first days of regrowth. Roots were the main net source of mobilized N. The quantitatively dominant N storage compounds were free amino acids. Free amino acids and soluble proteins in the roots decreased by 55 and 50%, respectively, and a substantial (approximately 38%) decrease in stubble protein was also observed. Although the relative abundance of several soluble proteins in roots decreased during the initial recovery from defoliation, no evidence was found for vegetative storage protein (VSP). Furthermore, rhizomes did not act as a N storage compartment. CONCLUSIONS: Production of new leaf area was entirely reliant, during the first week after defoliation, on N stores present in the plant. Mobilized N originated mainly from free amino acids and soluble proteins located in roots, and less so from proteins in stubble. Presence of VSP in the roots was not confirmed. The data suggest that rhizomes played an important role in N transport but not in N storage.     

Spatial and temporal changes in lipase activity sites during oil body mobilization in protoplasts from sunflower seedling cotyledons

Hydrolysis of triacylglycerols (TAGs) catalyzed by lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) action, is the principal biochemical event during oil body mobilization in germinating oilseeds. Employing a fluorescence microscopic technique developed in the author’s laboratory, a shift in the intracellular lipase activity has been demonstrated in the protoplasts of sunflower seedling cotyledons during seed germination. Lipase activity is primarily confined to protein storage vacuoles (PSVs) in 1 d old seedling cotyledons. At 2 d old stage, a relocalization of lipase activity begins and activity can be observed both on PSVs and oil bodies. At later stages of development (3–6 d), smaller PSVs coalesce into a large vegetative vacuole devoid of lipase activity. During this phase, lipase activity is confined to oil bodies only and maximum activity is detected in 4 d old seedlings, coinciding with maximum rate of lipolysis. Thus, present investigations on protoplasts from seedling cotyledons provide evidence for intracellular shift in lipase activity to sites of TAG hydrolysis (oil bodies) and also show a structural and functional reorganization of PSVs.     

肝细胞生长因子对骨髓内皮祖细胞的动员作用

目的: 分析肝细胞生长因子(HGF)能否动员骨髓内皮祖细胞,以及动员的内皮祖细胞能否参与创伤修复时的血管新生和内皮修复.方法: 将腺病毒HGF载体(adenovirus vector encoding HGF gene, Ad-HGF)经尾静脉注射到Balb/c小鼠体内,用ELISA方法检测血浆HGF水平的变化;用流式细胞术检测外周血CD34 细胞含量变化;对外周血单个核细胞进行分离、培养,并对生长的细胞克隆进行内皮细胞表面标志Tie-2、vW因子的免疫组化检测.建立雌性小鼠CCl4肝损伤模型,静脉移植HGF处理后雄性小鼠外周血单个核细胞到其体内,4 W后利用原位杂交技术检测新生肝组织中是否存在雄性细胞.结果: 注射Ad-HGF能明显提高小鼠血浆的HGF水平,并使外周血中以CD34、Tie-2和vW因子等为标志的内皮祖细胞的数量显著增多.这些细胞参与肝损伤修复时的血管新生.结论: HGF对骨髓内皮祖细胞具有明显的动员作用.     

Structural plasmid evolution as a result of coupled recombinations at<Emphasis Type="Italic"> bom</Emphasis> and<Emphasis Type="Italic"> cer</Emphasis> sites

We have studied the recombination of plasmids bearing bom and cer sites. The bom (basis of mobilization) site is required for conjugative transfer, while the cer (ColE1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.Communicated by W. Goebel     

Microbial biomass and activities associated with subsurface environments contaminated with chlorinated hydrocarbons

Soil microcosms and enrichment cultures from subsurface sediments and groundwaters contaminated with trichloroethylene (TCE) were examined. Total lipids, [I‐‘⁴C]acetate incorporation into lipids, and [Me‐³H]thymidine incorporation into DNA were determined in these subsurface environments. In heavily TCE‐contam‐inated zones (greater than 500 mg/L) radioisotopes were not incorporated into lipids or DNA. Radioisotope incorporation occurred in sediments both above and below the TCE plume. Phospholipid fatty acids (PLFA) were not detected, i.e., less than 0.5 pmol/L in heavily contaminated groundwater samples. In less contaminated waters, extracted PLFA concentrations were greater than 100 pmollL and microbial isolates were readily obtained. Degradation of 30–100 mg/L TCE was observed when sediments were amended with a variety of energy sources. Microorganisms in these subsurface sediments have adapted to degrade TCE at concentrations greater than 50 mg/L.     

Purification and characterisation of a P-selectin-binding molecule from the salivary glands of Ornithodoros moubata that induces protective anti-tick immune responses in pigs

Ornithodoros moubata is an argasid tick that lives in Africa in wild and synanthropic habitats. It feeds on warthogs, domestic swine and humans, and is able to transmit severe diseases such as human relapsing fever and African swine fever. The elimination of O. moubata from the synanthropic surroundings should improve control of the diseases it transmits. Previous attempts to develop a vaccine against O. moubata showed that a salivary gland extract (SGE) induced a protective response that inhibited the feeding of the ticks by up to 60%. Our aim in the present work was to isolate and characterise the salivary antigens responsible for the protective immune response induced with the SGE. The work reported here describes the finding and partial characterisation of a tick salivary glycoprotein of 44 kDa (Om44) that binds host P-selectin, presumably preventing the adhesion of leucocytes and platelets to vessel walls, thus allowing the ticks to complete their feeding. Preliminary analysis indicated that Om44 is not a homologue of the mammalian PSGL-1s and lacks sialyl-Lewis^X, Lewis^X and Lewis^Y determinants but carries heparin, which is the P-selectin-binding motif. Om44 is not recognised by the pig immune system after natural contact with O. moubata, but it can be neutralised by specific vaccine-induced antibodies, resulting in feeding inhibitions of around 50% in adults and nymphs-4, 25% in nymphs-3, 5% in nymphs-2 and 0% in nymphs-1 when they are fed on Om44-vaccinated pigs. In addition, the fecundity of females was inhibited by up to 43.8%. Om44 provides new prospects for the development of new anti-tick vaccines.     

Religious expression in public schools: Kirpans in Canada,hijab in France

The present study analyses the status of the Arab minority in Israel, as revealed in Project Renewal, one of the largest and most ambitious programmes implemented in the country during the 1980s. The analysis discloses how conflicts over symbolic issues, such as the definition of the collective's boundaries, found their way on to the Project Renewal agenda, and how they affected the project's dynamics. The article analyses the intensity of the conflicts evoked by the issue of citizen participation in the neighbourhood. It interprets the clashes over neighbourhood representation as essentially resulting from a core conflict, one in which the very boundaries of Israel's moral community appeared to be at stake. The conclusions are based on an analytical distinction between those components of the programme that may be viewed as related to liberal citizenship rights, and those pertaining to republican citizenship rights, that is, elements touching upon the definition of the boundaries of the social collective and the moral community.