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961.
Christopher P. Landowski Anne Huuskonen Ramon Wahl Ann Westerholm-Parvinen Anne Kanerva Anna-Liisa H?nninen Noora Salovuori Merja Penttil? Jari Natunen Christian Ostermeier Bernhard Helk Juhani Saarinen Markku Saloheimo 《PloS one》2015,10(8)
The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant. 相似文献
962.
963.
Sarah E. Gutowsky Yann Tremblay Michelle A. Kappes Elizabeth N. Flint John Klavitter Leona Laniawe Dan P. Costa Maura B. Naughton Marc D. Romano Scott A. Shaffer 《Ibis》2014,156(1):60-72
Past tracking studies of marine animals have primarily targeted adults, biasing our understanding of at‐sea habitat use toward older life stages. Anthropogenic threats persist throughout the at‐sea ranges of all life stages and it is therefore of interest to population ecologists and managers alike to understand spatiotemporal distributions and possible niche differentiation between age‐classes. In albatrosses, particularly little is known about the juvenile life stage when fledglings depart the colonies and venture to sea with no prior experience or parental guidance. We compared the dispersal of 22 fledgling Black‐footed Albatross Phoebastria nigripes between 2006 and 2008 using satellite telemetry and 16 adults between 2008 and 2009 using geolocaters from Midway Atoll National Wildlife Refuge, Northwest Hawaiian Islands. Following tag deployment, all fledglings spent several days within the calm atoll waters, then travelled northward until reaching 750–900 km from the colony. At this point, fledgling distributions approached the productive North Pacific Transition Zone (NPTZ). Rather than reaching the high chlorophyll a densities on the leading edge of this zone, however, fledglings remained in areas of low productivity in the subtropical gyre. In contrast, adult albatrosses from the same breeding colony did not utilize the NPTZ at this time of year but rather ranged throughout the highly productive northern periphery of the Pacific Ocean Basin among the shelf regions off Japan and the Aleutian Islands. The dichotomy in habitat use between fledglings and adults from Midway Atoll results in complete spatial segregation between age‐classes and suggests ontogenetic niche separation in this species. This research fills a large knowledge gap in at‐sea habitat use during a little known yet critical life stage of albatrosses, and contributes to a more comprehensive understanding of differential mortality pressure between age‐classes and overall conservation status for the vulnerable Black‐footed Albatross. 相似文献
964.
Marien J.C. Houtman Sanne M. Korte Yuan Ji Bart Kok Marc A. Vos Anna Stary-Weinzinger Marcel A.G. van der Heyden 《Biochemical and biophysical research communications》2014
Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6 μM) inward rectifier currents. 相似文献
965.
Biochemical reaction networks are subjected to large fluctuations attributable to small molecule numbers, yet underlie reliable biological functions. Thus, it is important to understand how regularity can emerge from noise. Here, we study the stochastic dynamics of a self-repressing gene with arbitrarily long or short response time. We find that when the mRNA and protein half-lives are approximately equal to the gene response time, fluctuations can induce relatively regular oscillations in the protein concentration. To gain insight into this phenomenon at the crossroads of determinism and stochasticity, we use an intermediate theoretical approach, based on a moment-closure approximation of the master equation, which allows us to take into account the binary character of gene activity. We thereby obtain differential equations that describe how nonlinearity can feed-back fluctuations into the mean-field equations to trigger oscillations. Finally, our results suggest that the self-repressing Hes1 gene circuit exploits this phenomenon to generate robust oscillations, inasmuch as its time constants satisfy precisely the conditions we have identified. 相似文献
966.
Siele Ceuppens Nico Boon Marc Heyndrickx Mieke Uyttendaele 《Journal of microbiological methods》2010,83(2):202-210
There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments. 相似文献
967.
968.
Cornelius F. H. Mueller Marc Ulrich Becher Sebastian Zimmer Sven Wassmann Barbara Keuler Georg Nickenig 《Molecular and cellular biochemistry》2010,333(1-2):261-267
The multidrug resistance-related protein-1 (MRP1) is important for the management of oxidative stress in vascular cells in vivo. Substrates of MRP1 are, among others, glutathione and the leukotriene C4 (LTC4), an eicosanoid and mediator of inflammation. Angiotensin (Ang) II infusion results in MRP1?/? mice compared to wild-type mice in improved endothelial function and reduced reactive oxygen species (ROS) formation. However, the interaction between Ang II, LTC4 and MRP1 is not completely understood and has never been investigated in vitro. Ang II induced in vascular smooth muscle cells (VSMC) the release of LTC4 and the generation of ROS. Pharmacologic inhibition of MRP1 via MK 571 significantly reduced Ang II-induced ROS release (L012-luminescence) in VSMC. The release of ROS after Ang II stimulation is inhibited, to a comparable degree, by blockade of the Cys-LT1 receptor with montelukast. Incubation of VSMC with recombined LTC4 and Ang II caused enhanced rates of proliferation in VSMC. This effect can be rescued by either MRP1 or Cys-LT1 receptor inhibition. Accordingly, stimulation of VSMC with LTC4 reduces intracellular levels of glutathione, but does not affect apoptosis. LTC4 stimulation results in a significant activation of MRP1, but does not alter MRP1 expression. These findings indicate a connection between Ang II, MRP1 and LTC4. Both, MRP1 and LTC4, are potentially promising targets for atheroprotective therapy. 相似文献
969.
Increase in seagrass distribution at Bourgneuf Bay (France) detected by spatial remote sensing 总被引:1,自引:0,他引:1
The changes in spatial distribution of intertidal Zostera noltii seagrass beds were studied with multispectral visible-infrared remote sensing in Bourgneuf Bay (France) over a 14-year period, between 1991 and 2005. Six SPOT satellite images acquired at low tide were calibrated using in situ spectroradiometric data and processed with the Normalized Difference Vegetation Index (NDVI). A steady and linear increase in meadow areas was observed between 1991 and 2005 with total surfaces colonized by Z. noltii increasing from 208 to 586 ha, respectively. A greater increase in the densest part of the meadow (NDVI > 0.4) was also observed: it represented only 15% of total meadow surfaces in 1991 vs. 35% in 2005. The seagrass expansion took place mainly towards the lower part of the intertidal zone, while in the upper intertidal zone the meadow appeared strictly limited by the +4 m (Lowest Astronomical Tide) bathymetric level. The influence of Z. noltii above-ground biomass variations on spectral reflectance was analyzed experimentally by spectrometry. Z. noltii displays a characteristic steep slope from 700 to 900 nm, increasing with increasing biomass. A quantitative relationship obtained experimentally between NDVI and the dry weight of leaves was used to produce a biomass distribution map. The distribution of Bourgneuf Bay intertidal seagrass beds is certainly constrained by the water turbidity and we suggest that tidal flat accretion could be a significant variable explaining the observed expansion downwards. With very limited spatial interactions, oyster aquaculture cannot be considered as a threat, while a recent increase in recreational hand fishing of Manila clams within the beds could become problematic. 相似文献
970.
Replication Protein A (RPA) is a single-stranded DNA-binding protein essential for DNA replication, repair, recombination and cell-cycle regulation. A human homolog of the RPA2 subunit, called RPA4, was previously identified and shown to be expressed in colon mucosal and placental cells; however, the function of RPA4 was not determined. To examine the function of RPA4 in human cells, we carried out knockdown and replacement studies to determine whether RPA4 can substitute for RPA2 in the cell. Unlike RPA2, exogenous RPA4 expression did not support chromosomal DNA replication and lead to cell-cycle arrest in G2/M. In addition, RPA4 localized to sites of DNA repair and reduced γ-H2AX caused by RPA2 depletion. These studies suggest that RPA4 cannot support cell proliferation but can support processes that maintain the genomic integrity of the cell. 相似文献