Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.
- MeSH
- Cryoelectron Microscopy MeSH
- Phosphorylation MeSH
- Humans MeSH
- Mitochondria * metabolism MeSH
- Polyphosphates * metabolism MeSH
- Protease La * metabolism MeSH
- Protein Domains MeSH
- Tyrosine * metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
(1R,2R-diaminocyclohexane)(dihydropyrophosphato) platinum(II), also abbreviated as RRD2, belongs to a class of potent antitumor platinum cytostatics called phosphaplatins. Curiously, several published studies have suggested significant mechanistic differences between phosphaplatins and conventional platinum antitumor drugs. Controversial findings have been published regarding the role of RRD2 binding to DNA in the mechanism of its antiproliferative activity in cancer cells. This prompted us to perform detailed studies to confirm or rule out the role of RRD2 binding to DNA in its antiproliferative effect in cancer cells. Here, we show that RRD2 exhibits excellent antiproliferative activity in various cancer cell lines, with IC50 values in the low micromolar or submicromolar range. Moreover, the results of this study demonstrate that DNA lesions caused by RRD2 contribute to killing cancer cells treated with this phosphaplatin derivative. Additionally, our data indicate that RRD2 accumulates in cancer cells but to a lesser extent than cisplatin. On the other hand, the efficiency of cisplatin and RRD2, after they accumulate in cancer cells, in binding to nuclear DNA is similar. Our results also show that RRD2 in the medium, in which the cells were cultured before RRD2 accumulated inside the cells, remained intact. This result is consistent with the view that RRD2 is activated by releasing free pyrophosphate only in the environment of cancer cells, thereby allowing RRD2 to bind to nuclear DNA.
- MeSH
- Cisplatin pharmacology MeSH
- Diphosphates pharmacology MeSH
- DNA metabolism MeSH
- Neoplasms * MeSH
- Organoplatinum Compounds pharmacology metabolism MeSH
- Oxaliplatin pharmacology MeSH
- Platinum pharmacology MeSH
- Antineoplastic Agents * pharmacology metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Pho91 is a vacuolar phosphate transporter that exports phosphate from the vacuolar lumen to the cytosol in yeast cells. In this study, we have demonstrated the pleiotropic effects of the PHO91 gene knockout in the methylotrophic yeast Ogataea parapolymorpha (Hansenula polymorpha, Ogataea angusta). The content of both acid-soluble and acid-insoluble inorganic polyphosphate (polyP) in the ∆pho91 cells was slightly higher compared to the strain with wild-type PHO91, when the cells were cultivated on glucose. The pho91-Δ mutations both in O. parapolymorpha and in Saccharomyces cerevisiae diminished resistance to cadmium and increased resistance to manganese and peroxide stresses. The cells of the mutant strain of O. parapolymorpha were unable to consume methanol due to the lack of methanol oxidase activity. We speculate that these effects are associated with the inability of mutant cells to mobilize phosphate from the vacuolar pool and/or defects in the signaling pathways involving phosphate, polyP, and inositol polyphosphates.
- MeSH
- Gene Knockout Techniques MeSH
- Polyphosphates * MeSH
- Publication type
- Journal Article MeSH
Podle definice života – množivost (replikace), metabolismus a energetická potřeba a schopnost reagovat na adekvátní podněty mají základní stavební molekuly života společnou hlavní nosnou strukturu: purinovou bázi – adenin, ribózu a dvě fosfátové skupiny. V tomto smyslu může molekula ATP představovat nosič a donor energie a organizátor molekulární výstavby, obnovy a replikace i signální molekulu v mechanismech reaktivity.
According to the definition of life – reproduction (replication), metabolism and energy requirements and the ability to respond to adequate stimuli, the basic building molecules of life have a common principal structure: the purine base - adenine, ribose and two phosphate groups. In this sense, the ATP molecule can represent a carrier and donor of energy and an organizer of molecular construction, renewal and replication, as well as a signalling molecule in reactivity mechanisms.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Newly designed hybrid biodegradable nanocomposite porous implant (HBNPI) modified with biogenic polyphosphate (bio-polyP) represents a very good opportunity to create bone fusion. Particularly effective utilization seems to be in spinal problems ,where the method of intervertebral fusion is mainly applied in injuries or degenerative spinal diseases. Our designed resorbable HBNPI composed of a hard ceramic outer ring and the inner soft collagen/nanohydroxyapatit foam modified with bio-polyP copy osteoconductive and osteoinductive properties of currently used autologous bone grafts. Synthetic HBNPI is prepared in laboratory manner eliminating both the complications associated with autografts collection and possibly the risk of infection transmission in the case of allografts. This project is focused on in-vivo testing of HBNPI ability to create intervertebral fusion in animal model and compare histology and ex-vivo bimechanics with standard autograft method. Results will create base of new and safer method of intervertebral fusion allowing subsequent clinical testing.
Nově navržený hybridní biodegradabilní nanokompozitní porézní implantát (HBNPI) modifikovaný biogenním polyfosfátem (bio-polyP) představuje velmi dobrou možnost jak vytvořit kostní fúzi. Zvláště efektivní využití se nabízí v páteřní problematice, kde při jejím poranění či degenerativním postižení je často využívána metoda tzv. intervertebrální dézy. Námi navržený resorbovatelný HBNPI složený z vnějšího tvrdého keramického prstence a vnitřní měkké kolagen/nanohydroxyapatitové pěny v kombinaci s bio-polyP kopíruje osteokonduktivní a osteoinduktivní vlastnosti dosud užívaných autologních kostních štěpů. Syntetický HBNPI je připravován laboratorně a tím odpadají komplikace spojené s odběrem autoštěpů, eventuálně i riziko přenosu infektu v případě užití aloštěpu. Tento projekt se zaměřuje na in-vivo testování schopnosti HBNPI vytvořit intevertebrální dézu na zvířecím modelu a provést histologické a ex-vivo biomechanické porovnání s dosud užívanými autoštěpy. Výsledky budou tvořit podklad k vytvoření nové a bezpečnější metodiky intervertebrální dézy a umožní následné klinické testování.
- MeSH
- Spinal Fusion methods MeSH
- Prosthesis Implantation MeSH
- Humans MeSH
- Models, Animal MeSH
- Nanocomposites therapeutic use MeSH
- Spine surgery MeSH
- Polyphosphates therapeutic use MeSH
- Swine MeSH
- Materials Testing MeSH
- Absorbable Implants MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Evaluation Study MeSH
- Conspectus
- Ortopedie. Chirurgie. Oftalmologie
- NML Fields
- ortopedie
- technika lékařská, zdravotnický materiál a protetika
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.
- MeSH
- Cell Wall chemistry MeSH
- Cell Cycle * MeSH
- Time Factors MeSH
- Chlorophyta cytology growth & development MeSH
- Guanine analysis MeSH
- Lipid Droplets metabolism MeSH
- Lipids analysis MeSH
- Microscopy * MeSH
- Polyphosphates analysis MeSH
- Spectrum Analysis, Raman * MeSH
- Starch analysis MeSH
- Cell Size MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
The green algal genus Cylindrocystis is widespread in various types of environments, including extreme habitats. However, very little is known about its diversity, especially in polar regions. In the present study, we isolated seven new Cylindrocystis-like strains from terrestrial and freshwater habitats in Svalbard (High Arctic). We aimed to compare the new isolates on a molecular (rbcL and 18S rDNA), morphological (light and confocal laser scanning microscopy), and cytological (Raman microscopy) basis. Our results demonstrated that the Arctic Cylindrocystis were not of a monophyletic origin and that the studied strains clustered within two clades (tentatively named the soil and freshwater/glacier clades) and four separate lineages. Morphological data (cell size, shape, and chloroplast morphology) supported the presence of several distinct taxa among the new isolates. Moreover, the results showed that the Arctic Cylindrocystis strains were closely related to strains originating from the temperate zone, indicating high ecological versatility and successful long-distance dispersal of the genus. Large amounts of inorganic polyphosphate (polyP) grains were detected within the chloroplasts of the cultured Arctic Cylindrocystis strains, suggesting effective luxury uptake of phosphorus. Additionally, various intracellular structures were identified using Raman microscopy and cytochemical and fluorescent staining. This study represents the first attempt to combine molecular, morphological, ecological, and biogeographical data for Arctic Cylindrocystis. Our novel cytological observations partially explain the success of Cylindrocystis-like microalgae in polar regions.
- MeSH
- Chlorophyta * MeSH
- Phylogeny MeSH
- Polyphosphates MeSH
- Streptophyta * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Arctic Regions MeSH
- Svalbard MeSH
The moving volutin (polyphosphate) granules known as "dancing bodies" can be observed in the vacuoles of the yeast cells. The aim of work was to study the effects of cultivation conditions and influences of physico-chemical factors on the motion of vacuolar volutin granules in Saccharomyces cerevisiae cells. The motion of granules is a non-Markovian process. It does not depend on the cell cycle phase, but depends on the growth stage. The maximal number of cells with "dancing bodies" was observed under cultivation of yeast at 25-28 °C and pH 5.4-5.8. Irradiation by non-ionizing electromagnetic radiation (EMR) of extremely high frequency (61.22 GHz, 100 μW, 30 min) had no effect on granule motion. After irradiation by non-ionizing EMR of very high frequency (40.68 MHz, 30 W, 30 min) the number of cells with "dancing bodies" decreased significantly and in 2 h restored almost to the control value. The possible nature of the moving volutin granules phenomenon due to metabolic processes is discussed.
- MeSH
- Electromagnetic Radiation MeSH
- Hydrogen-Ion Concentration MeSH
- Microscopy, Confocal MeSH
- Culture Media MeSH
- Image Processing, Computer-Assisted MeSH
- Motion MeSH
- Polyphosphates chemistry MeSH
- Saccharomyces cerevisiae chemistry cytology radiation effects MeSH
- Temperature MeSH
- Vacuoles chemistry radiation effects MeSH
- Publication type
- Journal Article MeSH
Cíl: Popsat nález neobvyklého močového konkrementu u pětiletého pacienta s kongenitální hypofosfatázií. Typ studie: Kazuistika se zhodnocením laboratorních nálezů. Název a sídlo pracoviště: Ústav lékařské chemie a klinické biochemie 2. lékařské fakulty Univerzity Karlovy a Fakultní nemocnice Motol, Pediatrická klinika 2. lékařské fakulty Univerzity Karlovy a Fakultní nemocnice Motol, V Úvalu 84, 150 06 Praha 5 Materiál a metody: Popis kazuistiky se zaměřením se především na analýzy močových konkrementů pomocí polarizační mikroskopie a infračervené spektrometrie. Výsledky: U pětiletého pacienta s kongenitální hypofosfatázií efektivně léčeného rekombinantní alkalickou fosfatázou (jejíž hodnota v séru představovala desetinásobek horní hranice referenčního rozmezí) byl prokázán raritní pyrofosfátový močový konkrement s příměsí sulfátu. K jeho tvorbě došlo v období, kdy byla u pacienta terapeuticky alkalizována moč (jejímž důvodem byla metafylaxe urátové urolitiázy), a pacient užíval profylakticky sulfonamidy před plánovanou litotrypsí. Závěr: Ačkoliv sérová koncentrace alkalické fosfatázy, jejíž funkcí je přeměna pyrofosfátu na fosfát, byla vysoká, byl u pacienta s kongenitální hypofosfatázií identifikován pyrofosfátový močový konkrement. Sulfátová složka konkrementu vznikla pravděpodobně na podkladě profylaktického podávání sulfonamidů.
Objectives: Identification of unusual renal pyrophosphate stone in 5 years old patient with congenital hypophosphatasia. Design: Case report and evaluation of laboratory results. Settings: Department of Medical Chemistry and Clinical Biochemistry, University Hospital Motol, Charles University, Second Faculty of Medicine, Department of Paediatrics, University Hospital Motol, Charles University, Second Faculty of Medicine, V Úvalu 84, 150 06 Prague 5 (Czech Republic). Material and methods: Case report focusing on determination of urinary stone composition by polarized light microscopy and infra-red spectrometry. Results: In 5 years old patient with effectively treated congenital hypophosphatasia by recombinant alkaline phosphatase (the serum value of which was ten times the upper limit of the reference range), rare pyrophosphate renal stone with addition of sulphate was demonstrated. The formation of the stone occurred in the period when the patient´s urine was therapeutically alkalized (due to the history of urate stones) and the patient used prophylactically sulfonamides prior to planned lithotripsy. Conclusion: Despite the high serum alkaline phosphatase concentration, the function of which is the conversion of pyrophosphate to phosphate, pyrophosphate renal stone was identified in a patient with congenital hypophosphatasia. The sulphate component of the stone is probably due to the prophylactic administration of sulfonamides.
- Keywords
- infračervená spektrometrie,
- MeSH
- Urinalysis methods MeSH
- Chemistry Techniques, Analytical methods MeSH
- Diphosphates analysis MeSH
- Hypophosphatasia * complications pathology MeSH
- Clinical Laboratory Techniques methods MeSH
- Kidney Calculi * etiology chemistry therapy MeSH
- Humans MeSH
- Lithotripsy methods MeSH
- Child, Preschool MeSH
- Sulfonamides therapeutic use MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Publication type
- Case Reports MeSH
