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21.
The process of translation termination in eukaryotes has been monitored by different types of assays, each with its own merits. We have developed an in vivo system where the reporter protein is secreted from the cells in culture thus enabling continuous monitoring of translation termination activity by simple sampling of the cell culture media. Using this system, cell cultures can be challenged with various stimuli during growth and the cellular responses on the translational level can be investigated in vivo as well as in vitro. Sampling is rapid, easy, and non-destructive to the cells, which enables measurement of translational fidelity in real time during time-course experiments. In particular with this system it is possible to assess very low levels of stop codon suppression. The reporter enzyme, secreted alkaline phosphatase (SEAP), becomes tagged with the S-peptide when there is readthrough of a stop codon placed between the C-terminus of the SEAP and the S-peptide. The tagged SEAP is bound to a matrix and the bound SEAP activity is measured versus total SEAP activity in the medium as a reference. With this assay we have confirmed that eRF1 acts as an antisuppressor in cells transfected with a cognate suppressor tRNA as well as in control cells, where a small but significant level of readthrough (suppression) could be detected. We have also characterized suppression of the three stop codons individually, and especially UGA is prone for wobbling. 相似文献
22.
Plant phosphorus uptake via external hyphae of arbuscular mycorrhizal fungi has been measured using compartmented systems
where a hyphal compartment is separated from a rooting compartment by a fine mesh. By labelling the soil within the hyphal
compartment with a radioactive phosphorus (P) isotope, hyphal uptake of P into the plant can be traced. The objective of this
growth chamber study was to test two hyphal compartments of different design with respect to their suitabilities for measurement
of hyphal P uptake. One hyphal compartment was simply a nylon mesh bag filled with 32P-labelled soil. The labelled soil in the other hyphal compartment was completely surrounded by an 8–10 mm layer of unlabelled
soil that served as a buffer zone. Mycorrhizal and non-mycorrhizal subterranean clover plants were grown in pots with a centrally
positioned hyphal compartment. Uptake of radioactive P by non-mycorrhizal control plants was 25% of that by mycorrhizal plants
with the mesh bag but only 3% when including the buffer zone. Based on this good control of non-mycorrhizal P uptake from
within the hyphal compartment and its greater ease of handling once produced, we judged the hyphal compartment including a
buffer zone to be superior to the mesh bag.
Accepted: 15 September 1998 相似文献
23.
Mandel U; Hassan H; Therkildsen MH; Rygaard J; Jakobsen MH; Juhl BR; Dabelsteen E; Clausen H 《Glycobiology》1999,9(1):43-52
Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc:
polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases).
Individual GalNAc-transferases appear to have different functions and
Northern analysis indicates that they are differently expressed in
different organs. This suggests that O-glycosylation may vary with the
repertoire of GalNAc-transferases expressed in a given cell. In order to
study the repertoire of GalNAc-transferases in situ in tissues and changes
in tumors, we have generated a panel of monoclonal antibodies (MAbs) with
well defined specificity for human GalNAc-T1, -T2, and -T3. Application of
this panel of novel antibodies revealed that GalNAc- transferases are
differentially expressed in different cell lines, in spermatozoa, and in
oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were
highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was
expressed in spermatozoa. The expression patterns in normal oral mucosa
were found to vary with cell differentiation, and for GalNAc-T2 and -T3
this was reflected in oral squamous cell carcinomas. The expression pattern
of GalNAc-T1 was on the other hand changed in tumors to either total loss
or expression in cytological poorly differentiated tumor cells, where the
normal undifferentiated cells lacked expression. These results demonstrate
that the repertoire of GalNAc-transferases is different in different cell
types and vary with cellular differentiation, and malignant transformation.
The implication of this is not yet fully understood, but it suggests that
specific changes in sites of O-glycosylation of proteins may occur as a
result of changes in the repertoire of GalNAc-transferases.
相似文献
24.
25.
The contribution of indigenous arbuscular mycorrhizal fungi (AMF) to growth and phosphorus (P) uptake by oilseed flax (Linum usitatissimum L.) was examined in two field experiments covering soil P levels from 20–86 mg kg-1 NaHCO3-extractable P. The fumigant dazomet was applied to the soil in half of the plots to obtain control plants with reduced mycorrhiza formation. An extensive AMF colonization of up to 48% of the root length was established in untreated soil of both experiments, although P fertilization reduced colonization to 28–39% at the latest harvests. Fumigation markedly decreased or totally prevented AMF colonization throughout the experiments. Root growth responded to fumigation by increased total and specific root length. Shoot P uptake was decreased by fumigation at soil P levels lower than ca. 50 mg kg-1 whereas shoot growth was reduced by fumigation at soil P levels lower than ca. 40 mg kg-1. The effects of fumigation were ascribed to the suppression of mycorrhiza formation. The effect of the AMF increased with decreasing soil P levels. Phosphorus inflow through roots (based on shoot P uptake) was reduced more strongly by fumigation than total P uptake. The P inflow through fungal tissue in roots was estimated to 4 × 10-14 mol P cm-1 s-1. We conclude that AMF are essential to flax growth at soil P levels below ca. 40 mg P kg-1, which is representative of the conditions under which most flax is grown. 相似文献
26.
27.
Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2. 总被引:1,自引:1,他引:0 下载免费PDF全文
G. F. Gao U. C. Gerth J. R. Wyer B. E. Willcox C. A. O'Callaghan Z. Zhang E. Y. Jones J. I. Bell B. K. Jakobsen 《Protein science : a publication of the Protein Society》1998,7(5):1245-1249
A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit. 相似文献
28.
Gunnhild Jakobsen Morten Engstrøm Ørnulf Paulsen Karin Sjue Sunil X. Raj Morten Thronæs Marianne Jensen Hjermstad Stein Kaasa Peter Fayers Pål Klepstad 《Trials》2018,19(1):707
Background
Despite the high prevalence of insomnia in patients with advanced cancer, there are no randomized controlled trials on pharmacological interventions for insomnia in this group of patients. A variety of pharmacological agents is recommended to manage sleep disturbance for insomnia in the general population, but their efficacy and safety in adults with advanced cancer are not established. Thus, there is a need to evaluate the effectiveness of medications for insomnia in order to improve the evidence in patients with advanced cancer. One of the most used sleep medications at present in patients with cancer is zopiclone.Methods
This is a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. A total of 100 patients with metastatic cancer who report insomnia will be randomly allocated to zopiclone or placebo. The treatment duration with zopiclone/placebo is 6 consecutive nights. The primary endpoint is patient-reported sleep quality during the final study night (night 6) assessed on a numerical rating scale of 0–10, where 0?=?Best sleep and 10?=?Worst possible sleep. Secondary endpoints include the mean patient-reported total sleep time and sleep onset latency during the final study night (night 6).Discussion
Results from this study on treatment of insomnia in advanced cancer will contribute to clinical decision-making and improve the treatment of sleep disturbance in this patient cohort.Trial registration
ClinicalTrials.gov, NCT02807922. Registered on 21 June 2016.29.
Sebastian Wacker Bjørn Mejdell Larsen Per Jakobsen Sten Karlsson 《Ecology and evolution》2018,8(16):8126-8134
Multiple paternity is an important characteristic of the genetic mating system and common across a wide range of taxa. Multiple paternity can increase within‐population genotypic diversity, allowing selection to act on a wider spectre of genotypes, and potentially increasing effective population size. While the genetic mating system has been studied in many species with active mating behavior, little is known about multiple paternity in sessile species releasing gametes into the water. In freshwater mussels, males release sperm into the water, while eggs are retained and fertilized inside the female (spermcast mating). Mature parasitic glochidia are released into the water and attach to the gills of fish where they are encapsulated until settling in the bottom substrate. We used 15 microsatellite markers to detect multiple paternity in a wild population of the freshwater pearl mussel (Margaritifera margaritifera). We found multiple paternity in all clutches for which more than two offspring were genotyped, and numbers of sires were extremely high. Thirty‐two sires had contributed to the largest clutch (43 offspring sampled). This study provides the first evidence of multiple paternity in the freshwater pearl mussel, a species that has experienced dramatic declines across Europe. Previous studies on other species of freshwater mussels have detected much lower numbers of sires. Multiple paternity in freshwater pearl mussels may be central for maintaining genetic variability in small and fragmented populations and for their potential to recover after habitat restoration and may also be important in the evolutionary arms race with their fish host with a much shorter generation time. 相似文献
30.
Hans-Christoph Steinhausen Helle Jakobsen Andrea Meyer Povl Munk J?rgensen Roselind Lieb 《PloS one》2016,11(1)