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11.
12.
Yvonne E. G. Eskildsen-Helmond Han A. A. Van Heugten Jos M. J. Lamers 《Molecular and cellular biochemistry》1996,157(1-2):39-48
There is now clear evidence that receptor-dependent phospholipase D is present in myocardium. This novel signal transduction pathway provides an alternative source of 1,2-diacylglycerol, which activates isoforms of protein kinase C. The members of the protein kinase C family respond differently to various combinations of Ca2+, phosphatidylserine, molecular species of 1,2-diacylglycerol and other membrane phospholipid metabolites including free fatty acids. Protein kinase C isozymes are responsible for phosphorylation of specific cardiac substrate proteins that may be involved in regulation of cardiac contractility, hypertrophic growth, gene expression, ischemic preconditioning and electrophysiological changes. The initial product of phospholipase D, phosphatidic acid, may also have a second messenger role. As in other tissues, the question how the activity of phospholipase D is controlled by agonists in myocardium is controversial. Agonists, such as endothelin-1, atrial natriuretic factor and angiotensin 11 that are shown to activate phospholipase D, also potently stimulate phospholipase C- in myocardium. PMA stimulation of protein kinase C inactivates phospholipase C and strongly activates phospholipase D and this is probably a major mechanism by which agonists that promote phosphatidyl-4,5-bisphosphate hydrolysis secondary activate phosphatidylcholine-hydrolysis. On the other hand, one group has postulated that formation of phosphatidic acid secondary activates phosphatidyl-4,5-bisphosphate hydrolysis in cardiomyocytes. Whether GTP-binding proteins directly control phospholipase D is not clearly established in myocardium. Phospholipase D activation may also be mediated by an increase in cytosolic free Ca2+ or by tyrosine-phosphorylation. 相似文献
13.
Alexander N. Glazer Yvonne M. Gindt Crystal F. Chan Kenneth Sauer 《Photosynthesis research》1994,40(2):167-173
Efficient production of ATP and NADPH by the light reactions of oxygen-evolving photosynthesis demands continuous adjustment of transfer of absorbed light energy from antenna complexes to Photosystem I (PS I) and II (PS II) reaction center complexes in response to changes in light quality. Treatment of intact cyanobacterial cells with N-ethylmaleimide appears to disrupt energy transfer from phycobilisomes to Photosystem I (PS I). Energy transfer from phycobilisomes to Photosystem II (PS II) is unperturbed. Spectroscopic analysis indicates that the individual complexes (phycobilisomes, PS II, PS I) remain functionally intact under these conditions. The results are consistent with the presence of connections between phycobiliproteins and both PS II and PS I, but they do not support the existence of direct contacts between the two photosystems.Abbreviations Chl
chlorophyll
- EPR
electron paramagnetic resonance
- NEM
N-ethylmaleimide
- PBS
phycobilisome
- PS
photosystem 相似文献
14.
The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F1 hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIIIirr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D0 of 1.4 Gy. In stages XIirr−Virr, when the cells were proliferatively active, the D0 was about 2.6 Gy. Based on the D0 values for sensitive and resistant spermatogonia and on the D0 for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing.
When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield (Y; in % abnormal cells) after a radiation dose D was described by Y = eτD, with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal eτD of 100. 相似文献
15.
Sven Göbel Karim E. Jaén Rita P. Fernandes Manfred Reiter Jennifer Altomonte Udo Reichl Yvonne Genzel 《Biotechnology and bioengineering》2023,120(11):3335-3346
The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 106 cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 108 TCID50/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs. 相似文献
16.
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
17.
High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed. 相似文献
18.
Human Microtubule-Associated Protein-2c Localizes to Dendrites and Axons in Fetal Spinal Motor Neurons 总被引:2,自引:0,他引:2
Joanna S. Albala Yvonne Kress Wan-Kyng Liu Karen Weidenheim Shu-Hui C. Yen Bridget Shafit-Zagardo 《Journal of neurochemistry》1995,64(6):2480-2490
Abstract: Microtubule-associated protein-2 (MAP-2) functions to maintain neuronal morphology by promoting the assembly of microtubules. MAP-2c is an alternately spliced form of MAP-2, containing the first 151 amino acids of high-molecular-weight (HMW) MAP-2 joined to the last 321 amino acids, eliminating 1,352 amino acids specific to HMW MAP-2. A polyclonal antibody generated to the splice site of human MAP-2c was used to determine its cellular localization. The MAP-2c antiserum was depleted of any HMW MAP-2 reactivity by absorption with HMW MAP-2 fusion protein. Western blot analysis of human fetal spinal cord homogenates demonstrated that the antibody is specific for human MAP-2c. MAP-2c immunoreactivity was found in the perinuclear cytoplasm and processes of anterior motor neurons and large processes of the posterior column in sections from 22–24-week human fetal spinal cord. Double-label confocal microscopy was performed using the MAP-2c polyclonal antibody and either a HMW MAP-2 or a neurofilament protein (highly phosphorylated 160- and 200-kDa protein) monoclonal antibody to identify these processes as dendrites or axons, respectively. HMW MAP-2 and MAP-2c colocalized in cell bodies and dendrites of anterior motor neurons, demonstrating for the first time the presence of native MAP-2c within dendrites. In addition, immunoelectron microscopy showed MAP-2c associated with microtubules in dendrites of motor neurons. MAP-2c and the neurofilament proteins were found in axons of the dorsal and ventral roots. The presence of MAP-2c within axons and dendrites suggests that MAP-2c contributes to neuronal plasticity during human fetal development. 相似文献
19.
Xavier Khawaja Non Evans Yvonne Reilly Christine Ennis Michael C. W. Minchin 《Journal of neurochemistry》1995,64(6):2716-2726
Abstract: The specific binding of [3H]WAY-100635 {N-[2-[4-(2-[O-methyl-3H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY-100635 association (k+1 = 0.069 ± 0.015 nM?1 min?1) and dissociation (k?1 = 0.023 ± 0.001 min?1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (Bmax) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY-100635 was ~36% higher compared with that of 8-hydroxy-2-(di-n-[3H]-propylamino)tetralin ([3H]8-OH-DPAT). The binding affinity of [3H]WAY-100635 was significantly lowered by the divalent cations CaCl2 (2.5-fold; p < 0.02) and MnCl2 (3.6-fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY-100635 binding to 5-HT1A receptors. The pharmacological binding profile of [3H]WAY-100635 was closely correlated with that of [3H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine1A (5-HT1A) sites in rat hippocampus. [3H]WAY-100635 competition curves with 5-HT1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites. 相似文献
20.
Yvonne M. van Houten Pam van Stratum Jan Bruin Alfred Veerman 《Entomologia Experimentalis et Applicata》1995,77(3):289-295
In Europe and North America the western flower thrips,Frankliniella occidentalis, is an important pest in various greenhouse crops, such as sweet pepper and cucumber. Two species of predatory mite are commercially
applied for biological control of this pest:Amblyseius cucumeris andA. barkeri. Thrips control is generally successful from March onwards. During winter, however, thrips control by these predatory mites
is less effective. An important reason for this is that the commercially applied strains of both mite species enter reproductive
diapause under short-day photoperiods, whereas the western flower thrips does not enter diapause. In this paper we report
on selection experiments for non-diapause in strains of both mite species, aimed at obtaining predators that do not enter
diapause under light- and temperature conditions prevailing in winter. Additional experiments were done to estimate the potential
of the selected lines as control agents ofF. occidentalis. Selection for non-diapause proved highly successful in both predatory mite species. In a New Zealand strain ofA. cucumeris diapause incidence decreased from 41% to 0% in about ten generations; in a Dutch strain ofA. barkeri diapause incidence decreased from 67% to 0% in about six generations. Furthermore, selection for non-diapause had no influence
on predator performance, measured as predation rate and oviposition rate on a diet of first instar thirps larvae. Rates of
predation and oviposition were the same for selected and unselected lines in both species; rates of predation and oviposition
were higher forA. cucumeris than forA. barkeri. After 18 months under non-diapause conditions, no less than 92% of a sample of the selected non-diapause line ofA. cucumeris did not enter diapause when tested under diapause-inducing conditions. This indicates that ‘non-diapause’ is a stable trait
in these predatory mites. Finally, a small-scale greenhouse experiment in a sweet pepper crop showed that the selected non-diapause
line ofA. cucumeris established successfully under diapause-inducing short-day conditions. 相似文献