To understand metabolic networks, fluxes and regulation, it is crucial to be able to determine the cellular and subcellular levels of metabolites. Methods such as PET and NMR imaging have provided us with the possibility of studying metabolic processes in living organisms. However, at present these technologies do not permit measuring at the subcellular level. The cameleon, a fluorescence resonance energy transfer (FRET)-based nanosensor uses the ability of the calcium-bound form of calmodulin to interact with calmodulin binding polypeptides to turn the corresponding dramatic conformational change into a change in resonance energy transfer between two fluorescent proteins attached to the fusion protein. The cameleon and its derivatives were successfully used to follow calcium changes in real time not only in isolated cells, but also in living organisms. To provide a set of tools for real-time measurements of metabolite levels with subcellular resolution, protein-based nanosensors for various metabolites were developed. The metabolite nanosensors consist of two variants of the green fluorescent protein fused to bacterial periplasmic binding proteins. Different from the cameleon, a conformational change in the binding protein is directly detected as a change in FRET efficiency. The prototypes are able to detect various carbohydrates such as ribose, glucose and maltose as purified proteins in vitro. The nanosensors can be expressed in yeast and in mammalian cell cultures and were used to determine carbohydrate homeostasis in living cells with subcellular resolution. One future goal is to expand the set of sensors to cover a wider spectrum of metabolites by using the natural spectrum of bacterial periplasmic binding proteins and by computational design of the binding pockets of the prototype sensors.
"A more accurate and sensitive technique to study metabolite compartmentalization is the use of FRET-based nanosensors (Figure 3C). These genetically encoded molecular nanosensors, with their prototype being developed for detecting calcium at nanomolar concentrations (Persechini et al., 1997), represent a new and promising technology to follow the spatial and temporal metabolic changes on the cellular and even subcellular level in living cells (Fehr et al., 2005; Lalonde et al., 2005). However, they do suffer from the severe limitation that each metabolite requires its own sensor protein for detection, thus making it essentially not applicable for true metabolomics. "
[Show abstract][Hide abstract] ABSTRACT: The main goal of metabolomics is the comprehensive qualitative and quantitative analysis of the time- and space-resolved distribution of all metabolites present in a given biological system. Because metabolite structures, in contrast to transcript and protein sequences, are not directly deducible from the genomic DNA sequence, the massive increase in genomic information is only indirectly of use to metabolomics, leaving compound annotation as a key problem to be solved by the available analytical techniques. Furthermore, as metabolites vary widely in both concentration and chemical behavior, there is no single analytical procedure allowing the unbiased and comprehensive structural elucidation and determination of all metabolites present in a given biological system. In this review the different approaches for targeted and non-targeted metabolomics analysis will be described with special emphasis on mass spectrometry-based techniques. Particular attention is given to approaches which can be employed for the annotation of unknown compounds. In the second part, the different experimental approaches aimed at tissue-specific or subcellular analysis of metabolites are discussed including a range of non-mass spectrometry based technologies.
The Plant Journal 04/2012; 70(1):39-50. DOI:10.1111/j.1365-313X.2012.04902.x · 5.97 Impact Factor
"Fluorescentlylabelled NIH 3T3 mouse fibroblast cells are dispersed into the well array by allowing a suspension of cells held above the collection to settle into the wells and adhere to the well bottom. The pattern of the cells populating the wells is determined by exciting the fluorescent cell membrane label at the appropriate wavelength (Okumoto et al., 2005). Once the location of the cells in the array has been determined, fluorescence measurements (of analyte) may then be made at other wavelengths. "
[Show abstract][Hide abstract] ABSTRACT: Nanotechnology research is rolling worldwide, having an impact on multiple sectors and with a general belief that
medical and biological applications will form the greatest area of expansion over the next decade. This field is mainly driven by an endeavour to bring radical solutions to areas of medical need, not fulfilled presently. This article discusses the basic concepts and developments in the field of nanosensors and their applications in pharmaceutical and medicine fields. Various types of nanosensors including optical nanosensors,
electrochemical nanosensors, chemical nanosensors, electrometers, biosensors, and deployable nanosensors are described. It describes the progression of this field of research from its birth up to the present, with emphasis on the techniques of sensor construction and their application to biomedical systems.
"As is shown in Figure 5, the cell population of all this treated plates is not significantly different after 24 h of incubation time but it is higher than control. One can deduce that D-ribose enters into the cell  and it is metabolized as carbon source increasing cell growth and consequently the cell number. Highest growth enhancement occurs at 0.05 mM K:D-Rib between 24 h and 48 h. "
[Show abstract][Hide abstract] ABSTRACT: The synergic action of KHCO3 and D-ribose is tested on A72 and HTB-126 cell lines proliferation using K:D-Rib solution. Altered Na+/K+ ATPase expression and activity were shown in patients with cancer. Studies in human epithelial-derived malignancies indicate that K+ depletion also occurs, contributing to the increased intracellular Na+/K+ ratio 1. D-ribose transformed to piruvate, enters into the Krebs's cycle and has a key role on energetic metabolism. The up-regulation of glycolysis in tumor cells is already well known and it is the rationale of F18-FDG PET diagnostic technique. D-ribose is synthesized by the non-oxidative transketolase PPP reaction.
Results with different K:D-Rib concentrations show that MTT salt interferes with K:D-Rib solution and therefore this method is not reliable. The UV/VIS measurements show that K:D-Rib solutions reduce MTT salt to formazan in absence of cells. Cell proliferation has then been evaluated analysing the digital photos of the Giemsa stained cells with MCID™ software. At 5 mM K:D-Rib concentration, the cell growth arrests between 48 h and 72 h; in fact the cell number after 48 h is around the same with respect to the control after 72 h. In case of HTB-126 human cancer cells, the growth rate was valuated counting the splitting times during 48 days: control cells were split sixteen times while 5 mM treated cells eleven times. Most relevant, the clonogenic assay shows that nine colonies are formed in the control cells while only one is formed in the 5 mM and none in 10 mM treated cells.
The K:D-Rib solution has an antioxidant behaviour also at low concentrations. Incubation with 5 mM K:D-Rib solution on A72 cells shows a cytostatic effect at 5 mM, but it needs more than 24 h of incubation time to evidence this effect on cell proliferation. At the same concentration on human HTB-126 cells, K:D-Rib solution shows a clear replication slowing but the cytostatic effect at 10 mM K:D-Rib solution only. Results on A72 cells indicate the K+ uptake could be determinant either to arrest or to slow down cell growth.
Cancer Cell International 08/2011; 11(1):30. DOI:10.1186/1475-2867-11-30 · 2.77 Impact Factor
Xi Xiang, Yuanjiao Tang, Qianying Leng, Lingyan Zhang, Li Qiu
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