spermine
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BACKGROUND: Fluorescence correlation spectroscopy (FCS) can be used for the determination of diffusion coefficients of single molecules. Since diffusion coefficients are correlated with size and shape of the labeled species, FCS provides information on conformational changes in plasmids aggregates. METHODS: A 10-kbp plasmid stained with PicoGreen was condensed by spermine or liposomes formulated from cationic lipid and egg phosphatidylcholine. RESULTS: The diffusion coefficient of DNA increases from 1.0 x 10(-12) m2/s to 3.2 x 10(-12) m2/s by the addition of spermine, whereas the addition of cationic liposomes leads to complexes characterized by diffusion coefficients with values ranging from 1.7 to 1.9 x 10(-12) m2/s. CONCLUSIONS: FCS experiments allow determining the diffusion coefficients of DNA-containing aggregates which provide information regarding the topology and homogeneity of the aggregate.
Photodynamic therapy (PDT) is one of the most promising methods of specific cancer treatment. However, commercially available photosensitizers (PSs) show significant drawbacks, such as side toxicity, low penetration ability, low blood solubility, low tumor selectivity etc. In addition, as was shown previously, a conjugation of polyamines with several toxic agents led to an increased toxicity to cancer cells. Here, we synthesized conjugates of two chlorine photosensitizers, purpurin 18 and pheophorbide a, with spermine in natural and Boc-protected form. Using specialized software, we calculated octanol-water partition coefficients for single protonation state (logP) of single PSs and PS/spermine conjugates. We found that the addition of spermine to chlorine PSs shifted the logP towards higher hydrophilicity in comparison to logP of single chlorines. In vitro studies on several cancer cells indicated that conjugation of purpurin 18 with spermine increased its retention in cancer cells. Using various concentrations of this conjugate, we found that lower concentrations (under 0.2μM) of purpurin 18/spermine conjugate launched apoptosis in HeLa cells. This combined with its high phototoxicity makes the purpurin 18/spermine conjugate a promising photosensitizer for PDT. Obtained results might serve as a basis for further studies of this potential third-generation PS on mammalian models in vivo.
- MeSH
- apoptóza účinky léků účinky záření MeSH
- chlorofyl analogy a deriváty chemie MeSH
- fotochemoterapie metody MeSH
- fotosenzibilizující látky chemie MeSH
- HeLa buňky MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- nádorové buněčné linie účinky léků účinky záření MeSH
- porfyriny chemie MeSH
- spermin chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The uptake of spermine into mammalian mitochondria indicated the need to identify its catabolic pathway in these organelles. Bovine liver mitochondria were therefore purified and their capacity for natural polyamine uptake was verified. A kinetic approach was then used to determine the presence of an MDL 72527-sensitive enzyme with spermine oxidase activity in the matrix of bovine liver mitochondria. Western blot analysis of mitochondrial fractions and immunogold electron microscopy observations of purified mitochondria unequivocally confirmed the presence of a protein recognized by anti-spermine oxidase antibodies in the mitochondrial matrix. Preliminary kinetic characterization showed that spermine is the preferred substrate of this enzyme; lower activity was detected with spermidine and acetylated polyamines. Catalytic efficiency comparable to that of spermine was also found for 1-aminododecane. The considerable effect of ionic strength on the Vmax/KM ratio suggested the presence of more than one negatively charged zone inside the active site cavity of this mitochondrial enzyme, which is probably involved in the docking of positively charged substrates. These findings indicate that the bovine liver mitochondrial matrix contains an enzyme belonging to the spermine oxidase class. Because H2O2 is generated by spermine oxidase activity, the possible involvement of the latter as an important signaling transducer under both physiological and pathological conditions should be considered.
- MeSH
- jaterní mitochondrie enzymologie MeSH
- játra enzymologie MeSH
- katalytická doména MeSH
- kinetika MeSH
- osmolární koncentrace MeSH
- oxidace-redukce MeSH
- oxidoreduktasy působící na CH-NH vazby antagonisté a inhibitory izolace a purifikace metabolismus MeSH
- peroxid vodíku metabolismus MeSH
- putrescin analogy a deriváty chemie MeSH
- skot MeSH
- spermidin metabolismus MeSH
- spermin metabolismus MeSH
- statická elektřina MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Supramolecular characteristics of two spermine amides of betulinic acid (1 and 2) were studied by measuring and evaluating their UV-VIS-NIR spectra in aqueous acetonitrile and DOSY-NMR spectra in tetradeuteromethanol, accompanied by atomic force microscopy (AFM) images, scanning electron microscopy (SEM) micrographs, and transmission electron microscopy (TEM) micrographs. Fibrous supramolecular self-assembly of 1 and 2 was observed by AFM images, as well as by the SEM and TEM micrographs. Bathochromic shifts of the absorbance maximum at 870nm to 1015-970nm in the UV-VIS-NIR spectra were observed with increasing water content in the acetonitrile/water systems, indicating formation of fibrous J-type aggregates. Variable temperature DOSY-NMR spectral measurement showed non-linear dependence that also suggests self-assembly behavior of the studied systems. Chiral supramolecular structures were formed by self-assembling due to the chirality of the monomeric molecules. Application of aqueous media during self-assembly procedures is an important factor in the development of targeted drug delivery systems.
The compaction of DNA plays a role in the nuclei of several types of cells and becomes important in the non-viral gene therapy. Thus, it is in the scope of research interest. It was shown, that spermine-induced compaction of large DNA molecules occurs in a discrete "all-or-non" regime, where the coexistence of free and folded DNA molecules was observed. In the case of intermediate-sized DNA molecules (approximately 10 kbp), so far, it was stated that the mechanism of folding is continuous. Here, we show, that neither a standard benchmark technique-dynamic light scattering, nor a single molecule technique such as fluorescence correlation spectroscopy, can decide what kind of mechanism is undertaken in the compaction process. Besides, we introduce an application of a new approach-fluorescence lifetime correlation spectroscopy. The method takes an advantage of a subtle lifetime change of an intercalating dye PicoGreen during the titration with spermine and based on that, it reveals the discrete mechanism of the process. Furthermore, we show that it allows for observation of the equilibrium state transition dynamics.
