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51.
Alexander Schouten Jan Roosien Fred A. van Engelen G. A. M. de Jong A. W. M. Borst-Vrenssen Jacoline F. Zilverentant Dirk Bosch Willem J. Stiekema Fred J. Gommers Arjen Schots Jaap Bakker 《Plant molecular biology》1996,30(4):781-793
The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed. 相似文献
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aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid 下载免费PDF全文
Ronald P. de Vries Charlotte H. Poulsen Susan Madrid Jaap Visser 《Journal of bacteriology》1998,180(2):243-249
An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. 相似文献
54.
Chris J. van Koppen Marcel W. Hermanussen Kiek N. Verrijp Jaap F. Rodrigues de Miranda Arie J. Beld Jan-Willem J. Lammers Cees A. M. van Ginneken 《Life sciences》1987,40(26):2561-2570
Specific binding of [125I]-(−)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (Kd=5.3±0.9 pmol/l and RT=78±7fmol/g tissue). The β1-selective antagonists atenolol and LK 203-030 inhibited specific [125I]-(−)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous β2-adrenoceptor population. In one subject using LK 203-030 a small β-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pKH- and pKL- values for the high and low affinity sites were 8.0±0.2 and 5.9±0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD2-value of 6.63±0.19. 相似文献
55.
Marthe C.J. Roex Charissa Wijnands Sabrina A.J. Veld Esther van Egmond Lisette Bogers Jaap J. Zwaginga Tanja Netelenbos Peter A. von dem Borne Hendrik Veelken Constantijn J.M. Halkes J.H. Frederik Falkenburg Inge Jedema 《Cytotherapy》2021,23(1):46-56
Background aimsTo reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) “to the bag” (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT.MethodsSixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from ≥9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation.ResultsIn vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) α/β T cells of 96.7% (range, 63.5–99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR α/β T-cell depletion. CD4pos T cells were depleted more efficiently than CD8pos T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR α/β T cells in the grafts after ALT incubation were not predictive for T-cell reconstitution or development of GVHD post- alloSCT.ConclusionsThe addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells. 相似文献
56.
Jolanda Tuma J. H. Strubbe Jan H. Strubbe Elisabeth Mocaë r Jaap M. Koolhaas 《Chronobiology international》2001,18(5):781-799
Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781-799, 2001) 相似文献
57.
Eli K. Moore Laura Villanueva Ellen C. Hopmans W. Irene C. Rijpstra Anchelique Mets Svetlana N. Dedysh Jaap S. Sinninghe Damsté 《Applied and environmental microbiology》2015,81(18):6333-6344
Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mosse peat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface. 相似文献
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59.
Multiple Sequence Alignment (MSA) methods are typically benchmarked on sets of reference alignments. The quality of the alignment can then be represented by the sum-of-pairs (SP) or column (CS) scores, which measure the agreement between a reference and corresponding query alignment. Both the SP and CS scores treat mismatches between a query and reference alignment as equally bad, and do not take the separation into account between two amino acids in the query alignment, that should have been matched according to the reference alignment. This is significant since the magnitude of alignment shifts is often of relevance in biological analyses, including homology modeling and MSA refinement/manual alignment editing. In this study we develop a new alignment benchmark scoring scheme, SPdist, that takes the degree of discordance of mismatches into account by measuring the sequence distance between mismatched residue pairs in the query alignment. Using this new score along with the standard SP score, we investigate the discriminatory behavior of the new score by assessing how well six different MSA methods perform with respect to BAliBASE reference alignments. The SP score and the SPdist score yield very similar outcomes when the reference and query alignments are close. However, for more divergent reference alignments the SPdist score is able to distinguish between methods that keep alignments approximately close to the reference and those exhibiting larger shifts. We observed that by using SPdist together with SP scoring we were able to better delineate the alignment quality difference between alternative MSA methods. With a case study we exemplify why it is important, from a biological perspective, to consider the separation of mismatches. The SPdist scoring scheme has been implemented in the VerAlign web server (http://www.ibi.vu.nl/programs/veralignwww/). The code for calculating SPdist score is also available upon request. 相似文献
60.
Rob C. van Lummel Stefan Walgaard Markus A. Hobert Walter Maetzler Jaap H. van Die?n Francisca Galindo-Garre Caroline B. Terwee 《PloS one》2016,11(3)