1. Cell-free extracts of all plants tested contained a novel enzyme activity (xyloglucan endotransglycosylase, XET) able to transfer a high-Mr portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide (Glc4Xyl3GalFuc; XG9). 2. A simple assay for the enzyme, using [3H]XG9 and based on the ability of the [3H]polysaccharide product to bind to filter paper, is described. 3. The enzyme was highly specific for xyloglucan as the glycosyl donor, and showed negligible transglycosylation of other polysaccharides, including CM-cellulose. 4. The Km for XG9 was 50 microM; certain other 3H-labelled xyloglucan oligosaccharides also acted as acceptors, and certain non-radioactive xyloglucan oligosaccharides competed with [3H]XG9 as acceptor; the minimum acceptor structure was deduced to be: [formula: see text] 5. The pH optimum was approx. 5.5 and the enzyme was less than half as active at pH 7.0. The enzyme was slightly activated by Ca2+, Mg2+, Mn2+, spermidine, ascorbate and 2-mercaptoethanol, and inhibited by Ag+, Hg2+, Zn2+ and La3+. 6. XET activity was essentially completely extracted by aqueous solutions of low ionic strength; Triton X-100, Ca2+, La3+, and Li+ did not enhance extraction. Negligible activity was left in the unextractable (cell-wall-rich) residue. 7. The enzyme differed from the major cellulases (EC 3.2.1.4) of pea in: (a) susceptibility to inhibition by cello-oligosaccharides, (b) polysaccharide substrate specificity, (c) inducibility by auxin, (d) requirement for salt in the extraction buffer and (e) activation by 2-mercaptoethanol. XET is therefore concluded to be a new enzyme activity (xyloglucan: xyloglucan xyloglucanotransferase; EC 2.4.1.-). 8. XET was detected in extracts of the growing portions of dicotyledons, monocotyledons (graminaceous and liliaceous) and bryophytes. 9. The activity was positively correlated with growth rate in different zones of the pea stem. 10. We propose that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that it thus causes the wall-loosening required for plant cell expansion.
Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24+/CD44+, CD133+, and aldehyde dehydrogenasebright) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.
1. A xyloglucan-derived nonasaccharide ([3H]XG9; Glc4,Xyl3,Gal,Fuc) was neither taken up by cultured plant cells nor appreciably hydrolysed by them, but a proportion of it became incorporated into extracellular polymers in all cultures tested (Spinacia, Daucus, Rosa, Acer, Capsicum, Zea and Festuca). 2. In Spinacia these polymers were soluble in 20% (w/v) trichloroacetic acid, had apparent Mr 20,000-30,000, were able to bind reversibly to cellulose powder and were susceptible to hydrolysis by endo-beta-(1----4)-D-glucanase, indicating that they were xyloglucans. 3. The linkage formed between [3H]XG9 and the xyloglucan was alkali-stable and glucanase-labile, indicating that the reaction responsible for the incorporation was a transglycosylation. 4. The reducing terminus of the XG9 moiety remained reducing (convertible into [3H]glucitol by NaBH4) after incorporation into the polymer, showing that the XG9 was the glycosyl acceptor and the polysaccharide the donor. 5. The results provide the first evidence that polymeric xyloglucans are subject in vivo to cleavage followed by transfer of the cut end on the other xyloglucan-related molecules. 6. Similar endotransglycosylation reactions could occur within the primary cell wall, between pairs of high-Mr structural xyloglucan molecules. Such a reaction would provide a mechanism for reversible wall loosening and may thus be relevant to our understanding of plant cell growth.
We propose computational empowerment as an approach and a Participatory Design response to challenges related to digitalization of society and the emerging need for digital literacy in K12 education. Our approach extends the current focus on computational thinking to include contextual, human-centred and societal challenges and impacts involved in students' creative and critical engagement with digital technology. Our research is based on the FabLab@School project, in which a PD approach to computational empowerment provided opportunities as well as further challenges for the complex agenda of digital technology in education. We argue that PD has the potential to drive a computational empowerment agenda in education by connecting political PD with contemporary visions for addressing a future digitalized labour market and society.
Natural killer (NK) cells are innate lymphocytes postulated to mediate resistance against primary haematopoietic but not solid tumor malignancies. Cancer stem cells (CSCs) are a small subset of malignant cells with stem-like properties which are resistant to chemo- and radiotherapies and are able to repopulate a tumor after cytoreductive treatments. We observed increased frequencies of stem-like tumor cells after irradiation, with increased expression of stress ligands on surviving stem-like cells. NK cells activated by low dose IL2 and IL15 displayed an increased ability to target solid tumor stem-like cells both and after irradiation. Mechanistically, both upregulation of stress-related ligands on the stem-like cells as well as debulking of non-stem populations contributed to these effects as determined by data from cell lines, primary tumor samples, and most relevant patient derived specimens. In addition, pretreatment of tumor-bearing mice with local radiation prior to NK transfer resulted in significantly longer survival indicating that radiation therapy in conjunction with NK cell adoptive immunotherapy targeting stem-like cancer cells may offer a promising novel radio-immunotherapy approach in the clinic.
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