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1.
《Molecular & cellular proteomics : MCP》2022,21(12):100438
Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy. 相似文献
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Colin K.W. Watts Robert L. Sutherland 《Biochemical and biophysical research communications》1984,120(1):109-115
Saturation and competitive binding analyses demonstrated the presence of a high affinity (KD = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver. 相似文献
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L. A. Nikitina 《Development genes and evolution》1978,184(2):109-113
Summary Experiments with oocyte enucleation and transplantation of germinal vesicles show that already at the beginning of the period of rapid growth, the oocyte karyoplasm contains the substances necessary for the appearance in the cytoplasm of the ability to divide. 相似文献
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The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate,
-lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane. 相似文献
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Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells). 相似文献
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Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana. 相似文献
9.
Legumin antibodies recognize polypeptides in coated vesicles isolated from developing pea cotyledons
Summary A highly enriched coated vesicle fraction has been isolated from cotyledons of developing pea seeds. This, and coated vesicles isolated from bovine brain as well as from bean leaves were subjected to SDS-PAGE followed by Western blotting with legumin antibodies. A distinct cross reaction with two polypeptides at around 60 kDa was seen, but only with the coated vesicles isolated from peas. Since legumin is synthesized as a 60 kDa precursor, but occurs as 40 and 20 kDa polypeptides in the protein body, we interpret our results as giving support to the idea that reserve proteins, like lysosomal proteins, are transported via coated vesicles.Abbreviations CV
coated vesicle
- DTT
dithiothreitol
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis 相似文献
10.
CMP-Neu5Gc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP-Neu5Gc enters the Golgi vesicles within 2 min; transport was saturable with high concentrations of the sugar-nucleotide and was dependent on temperature. A kinetic analysis gave an apparent Km of 1.3 μM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP-Neu5Ac, under the same incubation conditions. Furthermore, the uptake of CMP-Neu5Gc was inhibited by CMP-Neu5Ac, a substrate analogue. Conversely, the uptake of CMP-Neu5Ac was inhibited by CMP-Neu5Gc to the same extent, leading to the conclusion that the transport of CMP-Neu5Ac and CMP-Neu5Gc is mediated by the same carrier molecule. This transport system for CMP-Neu5Gc involves both CMP and CMP-Neu5Gc since intravesicular CMP induced the entry of external CMP-Neu5Gc. 相似文献