scholarly journals Ankylosis homologue (ANKH) controls extracellular citrate and pyrophosphate homeostasis and affects bone mechanical performance

2019 ◽  
Author(s):  
Flora Szeri ◽  
Stefan Lundkvist ◽  
Sylvia Donnelly ◽  
Udo F.H. Engelke ◽  
Kyu Rhee ◽  
...  
Keyword(s):  
Culture Medium ◽  
Mechanical Performance ◽  
Bone Development ◽  
Joint Space ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Human Patient ◽  
Inorganic Pyrophosphate ◽  
Normal Bone ◽  
Global Metabolite Profiling

AbstractThe membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of mineralization. Using global metabolite profiling, we identified citrate as the most prominent metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release of ATP and other nucleoside triphosphates. Ankank/ank mice, which lack functional ANK, had substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable in urine of a human patient lacking functional ANKH. Bone hydroxyapatite of Ankank/ank mice also contained markedly reduced levels of citrate and PPi and displayed diminished strength. Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis that is essential for normal bone development.

scholarly journals The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B

2021 ◽  
Vol 22 (3) ◽  
pp. 1175
Author(s):  
Ryuta Inukai ◽  
Kanako Mori ◽  
Keiko Kuwata ◽  
Chihiro Suzuki ◽  
Masatoshi Maki ◽  
...  
Keyword(s):  
Cell Death ◽  
Essential Gene ◽  
Susceptibility Gene ◽  
Target Protein ◽  
Hek293 Cells ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Endosomal Sorting ◽  
Cell Death Pathways ◽  
Apoptotic Protein

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

scholarly journals LRG1 Promotes Metastatic Dissemination of Melanoma through Regulating EGFR/STAT3 Signalling

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3279
Author(s):  
Yuet Ping Kwan ◽  
Melissa Hui Yen Teo ◽  
Jonathan Chee Woei Lim ◽  
Michelle Siying Tan ◽  
Graciella Rosellinny ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Melanoma Cell ◽  
Molecular Mechanisms ◽  
Complex Disease ◽  
Treatment Options ◽  
Melanoma Metastasis ◽  
Dependent Manner ◽  
Promoting Effect ◽  
Human Patient ◽  
Cell Invasiveness

Although less common, melanoma is the deadliest form of skin cancer largely due to its highly metastatic nature. Currently, there are limited treatment options for metastatic melanoma and many of them could cause serious side effects. A better understanding of the molecular mechanisms underlying the complex disease pathophysiology of metastatic melanoma may lead to the identification of novel therapeutic targets and facilitate the development of targeted therapeutics. In this study, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in melanoma development and progression. We first established the association between LRG1 and melanoma in both human patient biopsies and mouse melanoma cell lines and revealed a significant induction of LRG1 expression in metastatic melanoma cells. We then showed no change in tumour cell growth, proliferation, and angiogenesis in the absence of the host Lrg1. On the other hand, there was reduced melanoma cell metastasis to the lungs in Lrg1-deficient mice. This observation was supported by the promoting effect of LRG1 in melanoma cell migration, invasion, and adhesion. Mechanistically, LRG1 mediates melanoma cell invasiveness in an EGFR/STAT3-dependent manner. Taken together, our studies provided compelling evidence that LRG1 is required for melanoma metastasis but not growth. Targeting LRG1 may offer an alternative strategy to control malignant melanoma.

Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host–endophyte interaction in vitro and in vivo

2007 ◽  
Vol 53 (3) ◽  
pp. 380-390 ◽  
Author(s):  
Pious Thomas ◽  
Sima Kumari ◽  
Ganiga K. Swarna ◽  
T.K.S. Gowda
Keyword(s):  
Culture Medium ◽  
Carica Papaya ◽  
In Vitro Cultures ◽  
Dependent Manner ◽  
16S Rdna Sequence ◽  
16S Rdna Sequence Analysis ◽  
Single Organism ◽  
Dose Dependent

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (~1 cm) of papaya (Carica papaya L. ‘Surya’) planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2–4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea ( P. ananatis ), Enterobacter ( E. cloacae ), Brevundimonas ( B. aurantiaca ), Sphingomonas , Methylobacterium ( M. rhodesianum ), and Agrobacterium ( A. tumefaciens ) or two Gram-positive genera, Microbacterium ( M. esteraromaticum ) and Bacillus ( B. benzoevorans ) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550 = 0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.

scholarly journals Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

2011 ◽  
Vol 286 (22) ◽  
pp. 19215-19228 ◽  
Author(s):  
Frederic Cailotto ◽  
Pascal Reboul ◽  
Sylvie Sebillaud ◽  
Patrick Netter ◽  
Jean-Yves Jouzeau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Articular Chondrocytes ◽  
Specific Protein ◽  
Dependent Manner ◽  
Articular Chondrocyte ◽  
Experimental Conditions ◽  
Inorganic Pyrophosphate ◽  
Tgf Β1

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca2+. SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.

scholarly journals Glyoxalase I is a novel nitric-oxide-responsive protein

1999 ◽  
Vol 344 (3) ◽  
pp. 837-844 ◽  
Author(s):  
Atsushi MITSUMOTO ◽  
Kwi-Ryeon KIM ◽  
Genichiro OSHIMA ◽  
Manabu KUNIMOTO ◽  
Katsuya OKAWA ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Culture Medium ◽  
Molecular Mechanisms ◽  
Peptide Mapping ◽  
Glyoxalase I ◽  
Dependent Manner ◽  
Human Endothelial Cells ◽  
Native Form ◽  
No Donors ◽  
Dose Dependent

To clarify the molecular mechanisms of nitric oxide (NO) signalling, we examined the NO-responsive proteins in cultured human endothelial cells by two-dimensional (2D) PAGE. Levels of two proteins [NO-responsive proteins (NORPs)] with different pI values responded to NO donors. One NORP (pI 5.2) appeared in response to NO, whereas another (pI 5.0) disappeared. These proteins were identified as a native form and a modified form of human glyoxalase I (Glox I; EC 4.4.1.5) by peptide mapping, microsequencing and correlation between the activity and the isoelectric shift. Glox I lost activity in response to NO, and all NO donors tested inhibited its activity in a dose-dependent manner. Activity and normal electrophoretic mobility were restored by dithiothreitol and by the removal of sources of NO from the culture medium. Glox I was selectively inactivated by NO; compounds that induce oxidative stress (H2O2, paraquat and arsenite) failed to inhibit this enzyme. Our results suggest that NO oxidatively modifies Glox I and reversibly inhibits the enzyme's activity. The inactivation of Glox I by NO was more effective than that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), another NO-sensitive enzyme. Thus Glox I seems to be a novel NO-responsive protein that is more sensitive to NO than G3PDH.

scholarly journals Decrease in Membrane Fluidity and Traction Force Induced by Silica-Coated Magnetic Nanoparticles

2020 ◽  
Author(s):  
Tae Hwan Shin ◽  
Abdurazak Aman Ketebo ◽  
Da Yeon Lee ◽  
Seungah Lee ◽  
Seong Ho Kang ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Side Effects ◽  
Membrane Fluidity ◽  
Focal Adhesion ◽  
Membrane Damage ◽  
Cell Movement ◽  
Chemical Properties ◽  
Traction Force ◽  
Hek293 Cells ◽  
Dependent Manner

Abstract Background Nanoparticles are being used increasingly due to their unique physical and chemical properties and small size. It is well-known that nanoparticles cause side effects, however their biophysical assessment remains challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on the biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. Results Overdose (1.0 μg/µl) treatment of MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. During membrane damage, HEK293 cells were morphologically shrunk and aspect ratio of the cells were significantly decreased upon MNPs@SiO2(RITC) treatment. Each of traction force (measured in micropillar) was found to be increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, velocity of the cell movement was significantly decreased in MNPs@SiO2(RITC)-treated HEK293 cells. Moreover, intracellular ATP also decreased in a dose dependent manner upon MNPs@SiO2(RITC) treatment. To understand the biophysical changes in cells, we analysed transcriptome and metabolic profiles and generated metabotranscriptomics network. The network showed relationships among peroxidation of lipid, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipid and suppression of focal adhesion and cell movement.Conclusion Taken together, our results demonstrate that overdosage of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side effects.

scholarly journals The effects of 50 Hz magnetic field–exposed cell culture medium on cellular functions in FL cells

2019 ◽  
Vol 60 (4) ◽  
pp. 424-431 ◽  
Author(s):  
Yue Fei ◽  
Liling Su ◽  
Haifeng Lou ◽  
Chuning Zhao ◽  
Yiqin Wang ◽  
...  
Keyword(s):  
Cell Viability ◽  
Signaling Pathways ◽  
Relative Permittivity ◽  
Culture Medium ◽  
Biological Effects ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Cell Counting ◽  
International Agency ◽  
Dependent Manner

Abstract Although extremely low frequency magnetic fields (ELF-MFs) have been classified as a possible carcinogen for humans by the International Agency for Research on Cancer (IARC), their biological effects and underlying mechanisms are still unclear. Our previous study indicated that ELF-MF exposure influenced the relative permittivity of the saline solution, suggesting that the MF exposure altered physical properties of the solution. To explore the biophysical mechanism of ELF-MF–induced biological effects, this study examined the effects of 50 Hz sinusoidal MF at 0–4.0 mT on the permittivity of culture medium with phase-interrogation surface plasmon resonance (SPR) sensing. Then, the biological effects of MF pre-exposed culture medium on cell viability, the mitogen-activated protein kinase (MAPK) signaling pathways, oxidative stress, and genetic stabilities were analyzed using Cell Counting Kit-8, western blot, flow cytometry, γH2AX foci formation, and comet assay. The results showed that SPR signals were decreased under MF exposure in a time- and dose-dependent manner, and the decreased SPR signals were reversible when the exposure was drawn off. However, MF pre-exposed culture medium did not significantly change cell viability, intracellular reactive oxygen species level, activation of the MARK signaling pathways, or genetic stabilities in human amniotic epithelial cells (FL cells). In conclusion, our data suggest that the relative permittivity of culture medium was influenced by 50 Hz MF exposure, but this change did not affect the biological processes in FL cells.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

417 REPORTER GENE BIOASSAY OF FOLLICLE-STIMULATING HORMONE ACTIVITY IN MARE SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 365
Author(s):  
F. Sahmi ◽  
K. Sayasith ◽  
V. Portela ◽  
C. A. Price
Keyword(s):  
Cell Line ◽  
Granulosa Cell ◽  
Cell Lines ◽  
Reporter Gene ◽  
Colorimetric Assay ◽  
Animal Health ◽  
Major Effect ◽  
Hek293 Cells ◽  
Time Interval ◽  
Dependent Manner

Equine chorionic gonadotropin is secreted by the mare placenta and possesses both LH and FSH bioactivities in nonequine species. In ruminants, eCG is used commercially to induce superovulation. The production of commercial eCG is hampered by the variation in FSH and LH bioactivity of eCG between mares, and potentially results in batch-to-batch variation in eCG bioactivity. The objective of this study was to establish a cell-line-based bioassay of FSH activity in serum for use at eCG production facilities. Several cell lines were used for this study: HEK293 (kidney cells), KGN (a human granulosa cell line), and a new bovine granulosa cell line. The HEK293 and bovine granulosa cell lines did not express the FSH receptor (FSHR); therefore, the strategy was to cotransfect those cells with a FSHR expression plasmid and a cAMP reporter gene (β-galactosidase; β-Gal). The KGN cells transfected with β-Gal failed to respond to FSH and were not used further. The HEK293 and bovine cell lines responded to FSH in a dose-dependent manner, with a visible increase in β-Gal activity measured by colorimetric assay. The cells responded to eCG but not to LH, IGF1, or estradiol, demonstrating specificity for FSH activity. The minimum time of incubation required for clear bioactivity was 4 h. Activity was detected in serum from pregnant but not estrous mares. Attempts to create stable cell lines expressing both FSHR and β-Gal plasmids were not productive. We therefore attempted to create frozen batches of transiently transfected HEK293 cells. Several incubation conditions were tested and we succeeded in detecting β-Gal activity in response to eCG in thawed cells. The choice of serum during transfection had a major effect on the ability of the cells to respond to eCG after thawing, and the time interval between transfection and freezing significantly altered the magnitude of the response to eCG. The cells responded visibly to eCG treatment after 4-h incubation. In summary, we have developed a reasonably fast, colorimetric bioassay for FSH activity that can be used for serum in an on-farm setting. Supported by NSERC, AAFC, and Bioniche Animal Health.

scholarly journals Ultrastructural Features of Gold Nanoparticles Interaction with HepG2 and HEK293 Cells in Monolayer and Spheroids

Nanomaterials ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 2040 ◽  
Author(s):  
Boris Chelobanov ◽  
Julia Poletaeva ◽  
Anna Epanchintseva ◽  
Anastasiya Tupitsyna ◽  
Inna Pyshnaya ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Culture Medium ◽  
Hek293 Cells ◽  
Hydrodynamic Diameter ◽  
Multicellular Spheroids ◽  
Net Charge ◽  
Transmission Electron ◽  
Basal Plasma Membrane ◽  
Basal Plasma ◽  
Human Embryo Kidney

Use of multicellular spheroids in studies of nanoparticles (NPs) has increased in the last decade, however details of NPs interaction with spheroids are poorly known. We synthesized AuNPs (12.0 ± 0.1 nm in diameter, transmission electron microscopy (TEM data) and covered them with bovine serum albumin (BSA) and polyethyleneimine (PEI). Values of hydrodynamic diameter were 17.4 ± 0.4; 35.9 ± 0.5 and ±125.9 ± 2.8 nm for AuNPs, AuBSA-NPs and AuPEI-NPs, and Z-potential (net charge) values were −33.6 ± 2.0; −35.7 ± 1.8 and 39.9 ± 1.3 mV, respectively. Spheroids of human hepatocarcinoma (HepG2) and human embryo kidney (HEK293) cells (Corning ® spheroid microplates CLS4515-5EA), and monolayers of these cell lines were incubated with all NPs for 15 min–4 h, and fixed in 4% paraformaldehyde solution. Samples were examined using transmission and scanning electron microscopy. HepG2 and HEK2893 spheroids showed tissue-specific features and contacted with culture medium by basal plasma membrane of the cells. HepG2 cells both in monolayer and spheroids did not uptake of the AuNPs, while AuBSA-NPs and AuPEI-NPs readily penetrated these cells. All studied NPs penetrated HEK293 cells in both monolayer and spheroids. Thus, two different cell cultures maintained a type of the interaction with NPs in monolayer and spheroid forms, which not depended on NPs Z-potential and size.

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