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排序方式: 共有69条查询结果,搜索用时 31 毫秒
1.
This study shows that the zonula adhaerens in chicken retinal pigment epithelial (RPE) cells in situ consists of independent subunits which are composed of extracellular intermembrane discs sandwiched between cytoplasmic plaques. These zonula adhaerens complexes (ZACs) are hexagonally arranged within the junction. Previous immunocytochemical studies suggest that the zonula adhaerens region, composed of ZACs, contains the actin associated proteins vinculin and alpha-actinin. The intermembrane discs of ZACs likely mediate cell-to-cell adhesion whereas the cytoplasmic plaques are probably involved in binding the microfilaments of the relatively large circumferential microfilament bundles (CMBs), associated with the zonula adhaerens, to the cell membrane. The CMBs of chicken RPE cells in situ show striations similar to those found in stress fibers of other cell types and in CMBs of cultured epithelial cells. The observation that in the striated regions of CMBs the adjacent junctional membranes tend to follow an undulating path suggests that the CMBs are attached intermittently to the cell membrane and are contractile. The structural similarities between CMBs and stress fibers and the fact that they share similar actin associated proteins support the view that CMBs and stress fibers are related structures. 相似文献
2.
Litman GW; Rast JP; Shamblott MJ; Haire RN; Hulst M; Roess W; Litman RT; Hinds- Frey KR; Zilch A; Amemiya CT 《Molecular biology and evolution》1993,10(1):60-72
Immunoglobulins are encoded by a large multigene system that undergoes
somatic rearrangement and additional genetic change during the development
of immunoglobulin-producing cells. Inducible antibody and antibody-like
responses are found in all vertebrates. However, immunoglobulin possessing
disulfide-bonded heavy and light chains and domain-type organization has
been described only in representatives of the jawed vertebrates. High
degrees of nucleotide and predicted amino acid sequence identity are
evident when the segmental elements that constitute the immunoglobulin gene
loci in phylogenetically divergent vertebrates are compared. However, the
organization of gene loci and the manner in which the independent elements
recombine (and diversify) vary markedly among different taxa. One striking
pattern of gene organization is the "cluster type" that appears to be
restricted to the chondrichthyes (cartilaginous fishes) and limits
segmental rearrangement to closely linked elements. This type of gene
organization is associated with both heavy- and light-chain gene loci. In
some cases, the clusters are "joined" or "partially joined" in the germ
line, in effect predetermining or partially predetermining, respectively,
the encoded specificities (the assumption being that these are expressed)
of the individual loci. By relating the sequences of transcribed gene
products to their respective germ-line genes, it is evident that, in some
cases, joined-type genes are expressed. This raises a question about the
existence and/or nature of allelic exclusion in these species. The
extensive variation in gene organization found throughout the vertebrate
species may relate directly to the role of intersegmental
(V<==>D<==>J) distances in the commitment of the individual
antibody-producing cell to a particular genetic specificity. Thus, the
evolution of this locus, perhaps more so than that of others, may reflect
the interrelationships between genetic organization and function.
相似文献
3.
Sven Göbel Karim E. Jaén Marie Dorn Victoria Neumeyer Ingo Jordan Volker Sandig Udo Reichl Jennifer Altomonte Yvonne Genzel 《Biotechnology and bioengineering》2023,120(9):2639-2657
We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 106 cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 109 TCID50/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines. 相似文献
4.
João R. C. Ramos Alexander G. Rath Yvonne Genzel Volker Sandig Udo Reichl 《Biotechnology and bioengineering》2020,117(5):1533-1553
Mathematical modeling of animal cell growth and metabolism is essential for the understanding and improvement of the production of biopharmaceuticals. Models can explain the dynamic behavior of cell growth and product formation, support the identification of the most relevant parameters for process design, and significantly reduce the number of experiments to be performed for process optimization. Few dynamic models have been established that describe both extracellular and intracellular dynamics of growth and metabolism of animal cells. In this study, a model was developed, which comprises a set of 33 ordinary differential equations to describe batch cultivations of suspension AGE1.HN.AAT cells considered for the production of α1-antitrypsin. This model combines a segregated cell growth model with a structured model of intracellular metabolism. Overall, it considers the viable cell concentration, mean cell diameter, viable cell volume, concentration of extracellular substrates, and intracellular concentrations of key metabolites from the central carbon metabolism. Furthermore, the release of metabolic by-products such as lactate and ammonium was estimated directly from the intracellular reactions. Based on the same set of parameters, this model simulates well the dynamics of four independent batch cultivations. Analysis of the simulated intracellular rates revealed at least two distinct cellular physiological states. The first physiological state was characterized by a high glycolytic rate and high lactate production. Whereas the second state was characterized by efficient adenosine triphosphate production, a low glycolytic rate, and reactions of the TCA cycle running in the reverse direction from α-ketoglutarate to citrate. Finally, we show possible applications of the model for cell line engineering and media optimization with two case studies. 相似文献
5.
Highly oxidized protein aggregates accumulating in the brain during neurodegenerative diseases are often surrounded by microglia. Most of the microglial cells surrounding these plaques are activated and release a high amount of oxidizing species. In order to develop their toxic effects numerous oxidizing species need iron. To prevent this iron-dependent oxidation an iron-sequestering apparatus exists, including the major iron storage protein ferritin. Microglial cells damage their own protein pool during activation and it is still unknown whether microglial cells are able to maintain their iron-sequestering function during oxidative stress. Therefore, we explored the microglial cell line RAW to test the maintenance of ferritin under oxidizing conditions. Our investigations revealed a half-life of both ferritin chains of 3-3.5 h and a reduced half-life due to oxidation. This was due to the removal of oxidized ferritin by the proteasomal system. Ferritin de novo synthesis was also severely affected by oxidation. This results in a decreased ferritin pool due to acute oxidative stress. These data let us conclude that microglial cells do not increase their ferritin amount after oxidative stress and an increase in the iron storage capacity in these cells after treatment might be achieved only by a high iron saturation of the existing ferritin molecules. 相似文献
6.
Böhm E Sturm GJ Weiglhofer I Sandig H Shichijo M McNamee A Pease JE Kollroser M Peskar BA Heinemann A 《The Journal of biological chemistry》2004,279(9):7663-7670
Thromboxane (TX) A(2), a cyclooxygenase-derived mediator involved in allergic responses, is rapidly converted in vivo to a stable metabolite, 11-dehydro-TXB(2), which is considered to be biologically inactive. In this study, we found that 11-dehydro-TXB(2), but not the TXA(2) analogue U46,619 or TXB(2), activated eosinophils and basophils, as assayed by flow cytometric shape change. 11-Dehydro-TXB(2) was also chemotactic for eosinophils but did not induce, nor inhibit, platelet aggregation. Chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2) is an important chemoattractant receptor expressed by eosinophils, basophils, and TH2 lymphocytes, and prostaglandin (PG)D(2) has been shown to be its principal ligand. 11-Dehydro-TXB(2) induced calcium flux mainly from intracellular stores in eosinophils, and this response was desensitized after stimulation with PGD(2) but not other eosinophil chemoattractants. Shape change responses of eosinophils and basophils to 11-dehydro-TXB(2) were inhibited by the thromboxane (TP)/CRTH2 receptor antagonist ramatroban, but not the selective TP antagonist SQ29,548, and were insensitive to pertussis toxin. The phospholipase C inhibitor U73,122 attenuated both 11-dehydro-TXB(2)- and PGD(2)-induced shape change. 11-Dehydro-TXB(2) also induced the chemotaxis of BaF/3 cells transfected with hCRTH2 but not naive BaF/3 cells. At a threshold concentration, 11-dehydro-TXB(2) had no antagonistic effect on CRTH2-mediated responses as induced by PGD2. These data show that 11-dehydro-TXB(2) is a full agonist of the CRTH2 receptor and hence might cause CRTH2 activation in cellular contexts where PGD-synthase is not present. Given its production in the allergic lung, antagonism of the 11-dehydro-TXB(2)/CRTH2axis may be of therapeutic relevance. 相似文献
7.
Oxidative stress is supposed to play an important role in demyelinating diseases. Oligodendrocytes are the myelin-forming cells in the brain and are highly susceptible to oxidative stress due to their low antioxidative defense systems and high metabolic rate. In the present work, we tested the response of the oligodendrocyte cell line OLN 93 to oxidative stress. OLN 93 cell cultures are characterized by a loss of cell viability after oxidation. This loss of cell viability is accompanied by an increase in protein oxidation and consequently an elevated overall proteolysis. To minimize the oxidative damage, we tested the effects of the antioxidants alpha-lipoic acid and coenzyme Q(10). Both compounds were able to elevate cell viability and to decrease intracellular protein turnover and oxidant induced protein oxidation. Therefore, we concluded that the excessive oxidative damage of oligodendrocytes and their protein pool can be prevented by the usage of antioxidants. 相似文献
8.
Niklas J Priesnitz C Rose T Sandig V Heinzle E 《Applied microbiology and biotechnology》2012,93(4):1637-1650
Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing
that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α1-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed
in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well
as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different
feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture
with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed
a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of
the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the
higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one
hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production
of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased
with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the
regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for
further optimization of production cell lines and applied process conditions. 相似文献
9.
Verena Lohr Yvonne Genzel Ingo Jordan Dietmar Katinger Stefan Mahr Volker Sandig Udo Reichl 《BMC biotechnology》2012,12(1):1-9
Background
Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry.Results
Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay.Conclusions
A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis. 相似文献10.
This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth. 相似文献