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1.
《Journal of molecular biology》2021,433(17):166665
Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells. 相似文献
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Bone morphogenetic protein 2 (BMP-2) has been known for decades as a strong osteoinductive factor and for clinical applications is combined solely with collagen as carrier material. The growing concerns regarding side effects and the importance of BMP-2 in several developmental and physiological processes have raised the need to improve the design of materials by controlling BMP-2 presentation. Inspired by the natural cell environment, new material surfaces have been engineered and tailored to provide both physical and chemical cues that regulate BMP-2 activity. Here we describe surfaces designed to present BMP-2 to cells in a spatially and temporally controlled manner. This is achieved by trapping BMP-2 using physicochemical interactions, either covalently grafted or combined with other extracellular matrix components. In the near future, we anticipate that material science and biology will integrate and further develop tools for in vitro studies and potentially bring some of them toward in vivo applications. 相似文献
4.
Hongchi Yu Yang Shen Jingsi Jin Yingying Zhang Tang Feng 《Cell Adhesion & Migration》2018,12(1):56-68
Hepatocellular carcinoma (HCC) is a subtype of malignant liver cancer with poor prognosis and limited treatment options. It is noteworthy that mechanical forces in tumor microenvironment play a pivotal role in mediating the behaviors and functions of tumor cells. As an instrumental type of mechanical forces in vivo, fluid shear stress (FSS) has been reported having potent physiologic and pathologic effects on cancer progression. However, the time-dependent mechanochemical transduction in HCC induced by FSS remains unclear. In this study, hepatocellular carcinoma HepG2 cells were exposed to 1.4 dyn/cm2 FSS for transient duration (15s and 30s), short duration (5 min, 15 min and 30 min) and long duration (1h, 2h and 4h), respectively. The expression and translocation of Integrins induced FAK-Rho GTPases signaling events were examined. Our results showed that FSS endowed HepG2 cells with higher migration ability via reorganizing cellular F-actin and disrupting intercellular tight junctions. We further demonstrated that FSS regulated the expression and translocation of Integrins and their downstream signaling cascade in time-dependent patterns. The FSS downregulated focal adhesion components (Paxillin, Vinculin and Talin) while upregulated the expression of Rho GTPases (Cdc42, Rac1 and RhoA) in long durations. These results indicated that FSS enhanced tumor cell migration through Integrins-FAK-Rho GTPases signaling pathway in time-dependent manners. Our in vitro findings shed new light on the role of FSS acting in physiologic and pathological processes during tumor progression, which has emerged as a promising clinical strategy for liver carcinoma. 相似文献
5.
Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules 总被引:2,自引:0,他引:2
Burkhard Schlosshauer 《Journal of neurochemistry》1989,52(1):82-92
To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules. 相似文献
6.
《Developmental cell》2022,57(14):1694-1711.e7
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7.
A range of surfactants, including the anionic sodium dodecyl sulfate, the cationic cetyltrimethylammonium bromide and the nonionics octylphenoxy polyethoxyethanol (Triton X100) and polyoxyethylene 20 sorbitan monoleate (Tween 80) was studied for effects on proliferation, contractibility and attachment of cultured human fibroblasts. Only ionic surfactants exhibited a stimulatory effect on fibroblast proliferation, whereas all the surfactants tested increased the contraction of collagen gels containing fibroblasts, with the greatest effect from the non-ionic surfactants. This activity was not correlated with an increase of cell population or cell attachment within the collagenous matrix. The activity of the surfactants was seen only at levels close to their LD50 values and in a narrow range of concentrations. Thus, we consider that they are the result of the so-called hormesis phenomenon.Abbreviations CTAB
cetyltrimethyl-ammonium bromide
- FCS
fetal calf serum
- LD100
dose lethal to 100% of exposed
- MCD
maximal contraction dose
- PDL
population doubling level
- SDS
sodium dodecyl sulfate 相似文献
8.
J. Boonjawat P. Chaisiri J. Limpananont S. Soontaros P. Pongsawasdi S. Chaopongpang S. Pornpattkul B. Wongwaitayakul L. Sangduan 《Plant and Soil》1991,137(1):119-125
In non-legumes associative nitrogen-fixing system, several genera of rhizobacteria have been reported. The object of this paper is to summarize the current understanding of how rhizobacteria adhere to the root surface of non-legumes especially rice and other cereal crops. Evidence for involvement of rice lectin in adhesion will be reviewed. An emphasis will be placed on theKlebsiella R15 ammonium assimilation system in free-living state and in associative state with rice seedlings. Nitrogenase and glutamine synthetase (GS) activities of associativeKlebsiella increased significantly in the rhizosphere of rice comparing to the free-living state. In rice, the soluble form of GS specific activity appear to be slightly lower than in rice root in the absence of bacteria. These results suggest that nitrogen-fixing activity has been enhanced during association. The dinitrogen fixed should be changed to amino acids via GS-GOGAT pathway in bacteria. Transfer of fixed nitrogen and assimilation in the rice plant is the problem that needs to be solved in order to improve the efficiency of associative nitrogen fixation. 相似文献
9.
Michele Solem Angela Helmrich Paul Collodi David Barnes 《Molecular and cellular biochemistry》1991,100(2):141-149
Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis. 相似文献
10.
Philip Skehan James E. Thomas Susan J. Friedman 《In vitro cellular & developmental biology. Plant》1986,22(11):632-636
Summary Sarcoma 180 monolayers spontaneously shed single cells and small multicellular aggregates into the surrounding medium to produce
a dual population of floating and substratum-attached cells. Shedding was a motility-associated event that occurred when cells
attempted to migrate over one another. It resulted from a combination of cell shape change and active motility, which increased
sensitivity to fluid shear dislodgement by reducing a cell's surface area of adhesive contact and increasing strain tension
at its adhesive contact points. Shedding occurred at all phases of the cell cycle. Extracellular matrix but not conditioned
medium enhanced the floating subpopulation by slowing the kinetics of rattachment to plastic and cellular substrata. Although
sarcoma 180 cells are anchorage independent in the sense that they grow readily in single cell suspension, they nevertheless
exhibited anchorage modulation of their cell cycle. Short periods in suspension produced a mild G1 accumulation, whereas longer periods of anchorage deprivation led to a mild G2 accumulation which appeared to result from an interference with cytokinesis.
This work was supported by grants from the Medical Research Council of Canada, The National Cancer Institute of Canada, the
Alberta Heritage Savings and Trust Fund for Applied Cancer Research, and the Alberta Heritage Fund for Medical Research. 相似文献