1,720,989 research outputs found
Simultaneous imaging of cytosolic and mitochondrial Ca2+ concentration during glucose-induced oscillations in single primary mouse beta-cells.
No full textCHIMERIC PROTEINS FOR MEASURING ATP CONCENTRATIONS IN PERICELLULAR SPACE AND RELATED SCREENING METHOD
No full textThe invention relates to luminescent chimeric proteins comprising a first N-terminal protein sequence, a second protein sequence and a third C-terminal protein sequence wherein: (i) said first and said third protein sequence are a leader sequence and an anchor sequence belonging to at least a receptor localized on a plasma membrane site; (ii) said second protein sequence encodes for the full-length or partial sequence of a photoprotein and is inserted in frame between said first and said third sequence (i); said chimeric protein being addressed to said plasma membrane site of the cell wherein it is expressed
Stable expression in HEK293 cells and characterization of P2X7 isoform B: new hints on P2X7 mediated cell survival.
No full textA new luciferase target to the extracellular surface of the plasma membrane reveals a new pathway for ATP release in live intact cells.
No full textCharacterization of P2X7 isoform B sheds light on the role of pore versus channel activity in P2X7 mediated life and death.
No full textP2X7 isoform B (Gene bank AY847299, P2X7B) is a naturally occurring splice variant of P2X7 full-length receptor (Gene
Bank accession n NT-009775, P2X7A). P2X7B mRNA retains introns between exons 10 and 11 thus causing the loss of the
C terminal tail and incorporation of 18 amminoacids. If compared to P2X7A, P2X7B variant shows a reduced activity as
ion channel and a complete loss of the pore forming ability.
We engineered and stably expressed P2X7B in HEK293 cells were we could confirm that its activity is restricted to that of
small anion channel. P2X7B also loose P2X7A ability of ATP release in the extracellular milieu.
Nevertheless, P2X7B expression confers to HEK293 cells a longer survival in the absence of serum. P2X7B transfectants
also gain an increased competence in growth and migration in serum free soft agar, suggesting a possible role of the receptor
in facilitating tissue infiltration.
This increased resistance to unfavorable colture conditions is dependent on extracellular ATP, as treatment with apyrase
significantly reduces it. P2X7B transfectants show an higher content of intracellular ATP if compared to mock cells,
suggesting an increased metabolic activity. This hypothesis is also supported by a greater content of reticular calcium in
P2X7B cells and by an increased nuclear translocation of the calcineurin activated nuclear factor NFATc1. Moreover, in the
same cells there is and loss of Bz-ATP evoked cell blebbing.
It is tempting to speculate that while P2X7B expression will represent a proliferative advantage for the cell its counterpart,
P2X7A, will also expose it to death. These different phenotypes will be dependent on channel versus pore forming activity
A new method for measuring local ATP release at the extracellular surface of live intact cells
No full textSurface plasmon resonance analysis to detect the β+ IVSI-110 thalassemia mutation in circulating cell-free fetal DNA
No full textIn this manuscript we demonstrated Surface-Plasmon Resonance (SPR) technology is able to identify a thalassemia point mutation in cffDNA obtained from plasma of pregnant women
Real time detection of extracellular ATP in tumor microenvironment.
No full textBackground: The tumor microenvironment plays a critical role in tumor initiation, progression and invasion. Nucleotides,
such as ATP, have a surprisingly wide range of modulatory effects on tumor cells, however techniques for measuring ATP in
the extracellular environment are still rudimentary. Here we show an in vivo method for measuring extracellular ATP release
in tumor microenvironment.
Methods: We stably expressed pmeLUC, a chimeric luciferase targeted to the extracellular side of the plasma membrane, in
HEK293 cell lines. A stable cell clone (HEK293-pmeLUC) was then injected anesthetized nude/nude mice bearing or not a
tumor. Bioluminescence was then monitored by IVIS imaging system (Xenogen).
Results: Endovenous, intraperitoneal and subcutaneous inoculation of HEK293-pmeLUC cells in healthy nude mice caused
a modest, barely detectable, increase in luminescence. On the contrary, bioluminescence observed after injection of the
probe into mice bearing the OVCAR-3 human ovarian carcinoma or the MZ2-MEL human melanoma increased strongly.
We then validated HEK293-pmeLUC cells as ATP reporters using the ATP-hydrolyzing enzyme, potato apyrase. Apyrase
injection cause a significative drop in luminescence. We then performed an in vitro calibration which revealed that ATP in
the tumor interstitium is in the hundrend micromolar range, while it is basically undetectable in healthy tissues. Our
approach offers a new tool for the investigation of the biochemical composition of tumor milieu and for development of
novel therapies based on the modulation of extracellular purine-based signalling
Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.
No full textBackgroundThere is growing awareness that tumour cells build up a "self-advantageous" microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.Methodology/principal findingsMeasurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.Conclusions/significanceOur results show that ATP in the tumour interstitium is in the hundreds micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling
SPR-based biosensor for non-invasive prenatal diagnosis of Y-chromosome
No full textSince the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, diagnostic non-invasive prenatal methods have been developed. As far as Y-chromosome identification, this might be important for therapeutic intervention on sex-associated pathologies such as Duchenne muscular dystrophy, hemophilia and congenital adrenal hyperplasia. Surface plasmon resonance (SPR)-based biosensors might be useful for these studies, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses.
In this study BiacoreTM X100 has been applied to identify the Y-chromosome in cffDNA obtained from plasma samples of 26 pregnant women at different gestational ages, analysing the binding between SRY-PCR products and an SRY-specific probe immobilized on the sensor chip. The results suggest that there is a statistically significant difference between samples collected by pregnancies carrying male or female fetus. Moreover cffDNA, obtained at early gestational ages (6th-7th week) and not detectable by conventional quantitative real-time PCR, can be discriminated with high accuracy and reliability using SPR-based biosensors (Breveglieri G et. al, Prenatal Diagnosis 2016, 36, 353–361) and pre-amplification steps. In conclusion these results should be considered as the basis of future developments for more extensive diagnostic trials
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