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991.
992.
The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO)
cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture
medium at various times after the initiation of transfection inhibited further cellular uptake of PEI–DNA particles. Using
this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio
of 2:1 (w/w) and a cell density of 2 × 106 cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the
rate and the level of PEI–DNA uptake in serum-free minimal medium were found to be dependent on the PEI–DNA ratio, the cell
density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated
transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian
cells for the purpose of large-scale transient recombinant protein production. 相似文献
993.
Deutsch GB Zielonka EM Coutandin D Weber TA Schäfer B Hannewald J Luh LM Durst FG Ibrahim M Hoffmann J Niesen FH Sentürk A Kunkel H Brutschy B Schleiff E Knapp S Acker-Palmer A Grez M McKeon F Dötsch V 《Cell》2011,144(4):566-576
TAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ~20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes. 相似文献
994.
The ability of the bacteriophage-encoded peptidoglycan hydrolases (endolysins) to destroy Gram-positive bacteria from without
makes these enzymes promising antimicrobials. Recombinant endolysins from Listeria monocytogenes phages have been shown to rapidly lyse and kill the pathogen in all environments. To determine optimum conditions regarding
application of recombinant Listeria phage endolysins in food or production equipments, properties of different Listeria endolysins were studied. Optimum NaCl concentration for the amidase HPL511 was 200 nM and 300 mM for the peptidases HPL118,
HPL500, and HPLP35. Unlike most other peptidoglycan hydrolases, all four enzymes exhibited highest activity at elevated pH
values at around pH 8–9. Lytic activity was abolished by EDTA and could be restored by supplementation with various divalent
metal cations, indicating their role in catalytic function. While substitution of the native Zn2+ by Ca2+ or Mn2+ was most effective in case of HPL118, HPL500, and HPLP35, supplementation with Co2+ and Mn2+ resulted in an approximately 5-fold increase in HPL511 activity. Interestingly, the glutamate peptidases feature a conserved
SxHxxGxAxD zinc-binding motif, which is not present in the amidases, although they also require centrally located divalent
metals for activity. The endolysins HPL118, HPL511, and HPLP35 revealed a surprisingly high thermostability, with up to 35%
activity remaining after 30 min incubation at 90°C. The available data suggest that denaturation at elevated temperatures
is reversible and may be followed by rapid refolding into a functional state. 相似文献
995.
Willecke F Zeschky K Ortiz Rodriguez A Colberg C Auwärter V Kneisel S Hutter M Lozhkin A Hoppe N Wolf D von zur Mühlen C Moser M Hilgendorf I Bode C Zirlik A 《PloS one》2011,6(4):e19405
Background
Strong evidence supports a protective role of the cannabinoid receptor 2 (CB2) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB2 receptor in Murine atherogenesis.Methods and Findings
Low density lipoprotein receptor-deficient (LDLR−/−) mice subjected to intraperitoneal injections of the selective CB2 receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB2 activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB2−/−/LDLR−/− mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB2+/+/LDLR−/− controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-illicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB2 receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro.Conclusion
Our study demonstrates that both activation and deletion of the CB2 receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB2 in other inflammatory processes. However, in the context of atherosclerosis, CB2 does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque. 相似文献996.
Leiprecht N Munoz C Alesutan I Siraskar G Sopjani M Föller M Stubenrauch F Iftner T Lang F 《Biochemical and biophysical research communications》2011,404(2):695-700
Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na+-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (Ig). Ig was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of Ig following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1. 相似文献
997.
Khalid E. M. Sedeek Giovanni Scopece Yannick M. Staedler Jürg Schönenberger Salvatore Cozzolino Florian P. Schiestl Philipp M. Schlüter 《Molecular ecology》2014,23(24):6192-6205
High pollinator specificity and the potential for simple genetic changes to affect pollinator attraction make sexually deceptive orchids an ideal system for the study of ecological speciation, in which change of flower odour is likely important. This study surveys reproductive barriers and differences in floral phenotypes in a group of four closely related, coflowering sympatric Ophrys species and uses a genotyping‐by‐sequencing (GBS) approach to obtain information on the proportion of the genome that is differentiated between species. Ophrys species were found to effectively lack postpollination barriers, but are strongly isolated by their different pollinators (floral isolation) and, to a smaller extent, by shifts in flowering time (temporal isolation). Although flower morphology and perhaps labellum coloration may contribute to floral isolation, reproductive barriers may largely be due to differences in flower odour chemistry. GBS revealed shared polymorphism throughout the Ophrys genome, with very little population structure between species. Genome scans for FST outliers identified few markers that are highly differentiated between species and repeatable in several populations. These genome scans also revealed highly differentiated polymorphisms in genes with putative involvement in floral odour production, including a previously identified candidate gene thought to be involved in the biosynthesis of pseudo‐pheromones by the orchid flowers. Taken together, these data suggest that ecological speciation associated with different pollinators in sexually deceptive orchids has a genic rather than a genomic basis, placing these species at an early phase of genomic divergence within the ‘speciation continuum’. 相似文献
998.
Wolf D Jehle F Ortiz Rodriguez A Dufner B Hoppe N Colberg C Lozhkin A Bassler N Rupprecht B Wiedemann A Hilgendorf I Stachon P Willecke F Febbraio M von zur Muhlen C Binder CJ Bode C Zirlik A Peter K 《PloS one》2012,7(3):e33026
Background
Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L – an established marker and mediator of cardiovascular disease – induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo.Methodology/Principal Findings
WT or CD40L−/− mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L−/− mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L−/− mice. However, CD40L−/− mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L−/− mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels.Conclusion
We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease. 相似文献999.
1000.
Mentor Sopjani Anja Kunert Kamil Czarkowski Fabian Klaus Jörg Laufer Michael Föller Florian Lang 《The Journal of membrane biology》2010,233(1-3):35-41
The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca2+ channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active S422DSGK1, constitutively active T308D,S473DPKB, wild-type PIKfyve, and S318APIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (ICa) resulting from TRPV6-induced Ca2+ entry and subsequent activation of Ca2+-sensitive endogenous Cl? channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes IH was increased by coexpression of S422DSGK1 and by T308D,S473DPKB. Coexpression of wild-type PIKfyve further increased IH in TRPV6 + S422DSGK1-expressing oocytes but did not significantly modify ICa in oocytes expressing TRPV6 alone. S318APIKfyve failed to significantly modify ICa in the presence and absence of S422DSGK1. S422DSGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6. 相似文献