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排序方式: 共有102条查询结果,搜索用时 31 毫秒
41.
Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation
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The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction. 相似文献
42.
Overproduction of the Escherichia coli recA protein without stimulation of its proteolytic activity. 总被引:19,自引:13,他引:6
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Plasmid pBEU14, which carries the Escherichia coli recA+ gene and which can be amplified by manipulation of growth temperature, was constructed. When pBEU14 deoxyribonucleic acid was amplified, a high rate of synthesis and accumulation of recA protein resulted. Amplification of the recA gene and protein did not cause induction of prophage lambda, indicating that the proteolytic activity of the recA protein was not stimulated. 相似文献
43.
The three-dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 A resolution to a crystallographic R-value of 16.5% with good stereo-chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2 s) but several parts of the molecule are distinctly different. The most significant differences from PLA2 s from bovine pancreas and rattlesnake occur in the stretches 56-80 and 85-89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The C alpha positions for residues 86-88 differ by about 6 A from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site. 相似文献
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Rehab Alnabhan Ahmed Gaballa Lisa-Mari Mörk Jonas Mattsson Michael Uhlin Isabelle Magalhaes 《Cytotherapy》2018,20(7):941-951
Background
The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum.Methods
T cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity.Results
CD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+.Conclusion
Our results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product. 相似文献46.
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49.
Ribonucleotide reductase is an indispensable enzyme for all cells, since it catalyses the biosynthesis of the precursors necessary for both building and repairing DNA. The ribonucleotide reductase class I enzymes, present in all mammals as well as in many prokaryotes and DNA viruses, are composed mostly of two homodimeric proteins, R1 and R2. The reaction involves long-range radical transfer between the two proteins. Here, we present the first crystal structure of a ribonucleotide reductase R1/R2 holocomplex. The biological relevance of this complex is based on the binding of the R2 C terminus in the hydrophobic cleft of R1, an interaction proven to be crucial for enzyme activity, and by the fact that all conserved amino acid residues in R2 are facing the R1 active sites. We suggest that the asymmetric R1/R2 complex observed in the 4A crystal structure of Salmonella typhimurium ribonucleotide reductase represents an intermediate stage in the reaction cycle, and at the moment of reaction the homodimers transiently form a tight symmetric complex. 相似文献
50.
A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation 总被引:1,自引:0,他引:1
Michael Uhlin Mantas Okas Jens Gertow Mehmet Uzunel Torkel B. Brismar Jonas Mattsson 《Cancer immunology, immunotherapy : CII》2010,59(3):473-477
Epstein–Barr virus (EBV)-related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications
after allogeneic stem cell transplantation and solid-organ transplantation. In immunosuppressed transplant recipients, the
activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary
treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the
donor or, more recently, third-party donors. However, both are time consuming and expensive and often it is too late to administer
cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific T cells, treated and cured
a patient suffering from PTLD with multiple-associated tissue lesions, using her haplo-identical mother as a donor. This treatment
approach paves way for a new possibility to within-days treat patients with life-threatening EBV-associated malignancies. 相似文献