Arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance in plants, whereas little is known regarding AMF contribution to sucrose and proline metabolisms under drought stress (DS). In this study, Funneliformis mosseae and Paraglomus occultum were inoculated into trifoliate orange (Poncirus trifoliata) under well watered and DS. Although the 71-days DS notably (P < 0.05) inhibited mycorrhizal colonization, AMF seedlings showed significantly (P < 0.05) higher plant growth performance and leaf relative water content, regardless of soil water status. AMF inoculation significantly (P < 0.05) increased leaf sucrose, glucose and fructose concentration under DS, accompanied with a significant increase of leaf sucrose phosphate synthase, neutral invertase, and net activity of sucrose-metabolized enzymes and a decrease in leaf acid invertase and sucrose synthase activity. AMF inoculation produced no change in leaf ornithine-δ-aminotransferase activity, but significantly (P < 0.05) increased leaf proline dehydrogenase activity and significantly (P < 0.05) decreased leaf both Δ1-pyrroline-5-carboxylate reductase and Δ1-pyrroline-5-carboxylate synthetase activity, resulting in lower proline accumulation in AMF plants under DS. Our results therefore suggest that AMF strongly altered leaf sucrose and proline metabolism through regulating sucrose- and proline-metabolized enzyme activities, which is important for osmotic adjustment of the host plant.

• MeSH
• fyziologický stres * MeSH
• Glomeromycota fyziologie   MeSH
• listy rostlin enzymologie   metabolismus   MeSH
• metabolismus sacharidů MeSH
• mykorhiza fyziologie   MeSH
• období sucha * MeSH
• počet mikrobiálních kolonií MeSH
• Poncirus růst a vývoj   metabolismus   mikrobiologie   MeSH
• prolin metabolismus   MeSH
• sacharosa metabolismus   MeSH
• semenáček metabolismus   MeSH
• voda metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• glukosa metabolismus   MeSH
• krysa rodu rattus MeSH
• rybí oleje metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the food's nutritional and technological properties. The rhizosphere of plants that accumulate fructopolysaccharides as inulin has been revealed as a source of filamentous fungi. These fungi can produce FOS either by inulin hydrolysis or by biosynthesis from sucrose, including unusual FOS with enhanced prebiotic properties. Here, we investigated the ability of Fusarium solani and Neocosmospora vasinfecta to produce FOS from different carbon sources. Fusarium solani and N. vasinfecta grew preferentially in inulin instead of sucrose, resulting in the FOS production as the result of endo-inulinase activities. N. vasinfecta was also able to produce the FOS 1-kestose and 6-kestose from sucrose, indicating transfructosylating activity, absent in F. solani. Moreover, the results showed how these carbon sources affected fungal cell wall composition and the expression of genes encoding for β-1,3-glucan synthase and chitin synthase. Inulin and fructose promoted changes in fungal macroscopic characteristics partially explained by alterations in cell wall composition. However, these alterations were not directly correlated with the expression of genes related to cell wall synthesis. Altogether, the results pointed to the potential of both F. solani and N. vasinfecta to produce FOS at specific profiles.

• MeSH
• fruktosa metabolismus   MeSH
• Fusarium * genetika   metabolismus   MeSH
• inulin * metabolismus   MeSH
• oligosacharidy MeSH
• sacharosa metabolismus   MeSH
• uhlík MeSH
• Publikační typ
• časopisecké články MeSH

Salinization of water and soil has a negative impact on tomato (Solanum lycopersicum L.) productivity by reducing growth of sink organs and by inducing senescence in source leaves. It has been hypothesized that yield stability implies the maintenance or increase of sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves through changes in sucrolytic enzymes and their regulation by phytohormones. In this study, classical and functional physiological approaches have been integrated to study the influence of metabolic and hormonal factors on tomato fruit sink activity, growth, and yield: (i) exogenous hormones were applied to plants, and (ii) transgenic plants overexpressing the cell wall invertase (cwInv) gene CIN1 in the fruits and de novo cytokinin (CK) biosynthesis gene IPT in the roots were constructed. Although salinity reduces fruit growth, sink activity, and trans-zeatin (tZ) concentrations, it increases the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) during the actively growing period (25 days after anthesis). Indeed, exogenous application of the CK analogue kinetin to salinized actively growing fruits recovered sucrolytic activities (mainly cwInv and sucrose synthase), sink strength, and fruit weight, whereas the ethylene-releasing compound ethephon had a negative effect in equivalent non-stressed fruits. Fruit yield was increased by both the constitutive expression of CIN1 in the fruits (up to 4-fold) or IPT in the root (up to 30%), owing to an increase in the fruit number (lower flower abortion) and in fruit weight. This is possibly related to a recovery of sink activity in reproductive tissues due to both (i) increase in sucrolytic activities (cwInv, sucrose synthase, and vacuolar and cytoplasmic invertases) and tZ concentration, and (ii) a decrease in the ACC levels and the activity of the invertase inhibitor. This study provides new functional evidences about the role of metabolic and hormonal inter-regulation of local sink processes in controlling tomato fruit sink activity, growth, and yield under salinity.

• MeSH
• biomasa MeSH
• buněčná stěna enzymologie   MeSH
• chlorid sodný farmakologie   MeSH
• cytokininy metabolismus   MeSH
• exprese genu MeSH
• geneticky modifikované rostliny MeSH
• invertasa genetika   metabolismus   MeSH
• kořeny rostlin účinky léků   genetika   metabolismus   MeSH
• květy účinky léků   genetika   metabolismus   MeSH
• kyselina abscisová metabolismus   MeSH
• ovoce účinky léků   genetika   metabolismus   MeSH
• regulátory růstu rostlin farmakologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sacharosa metabolismus   MeSH
• salinita MeSH
• sekvestrace uhlíku MeSH
• Solanum lycopersicum účinky léků   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

• MeSH
• biologické modely * MeSH
• buněčné dýchání MeSH
• citrátsynthasa metabolismus   MeSH
• kyslík metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mozek metabolismus   MeSH
• Sus scrofa MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Deuterium-depleted water (DDW) has a lower concentration of deuterium than occurs naturally (less than 145 ppm). While effects of DDW on cancer started to be intensively studied, the effects on cardiovascular system are completely unknown. Thus, we aimed to analyze the effects of DDW (55+/-5 ppm) administration to 12-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) treated with 15 % fructose for 6 weeks. Blood pressure (BP) and selected biochemical parameters were measured together with determination of nitric oxide synthase (NOS) activity and iNOS and eNOS protein expressions in the left ventricle (LV) and aorta. Neither DDW nor fructose had any significant effect on BP in both strains. DDW treatment decreased total cholesterol and triglyceride levels in WKY, but it was not able to prevent increase in the same parameters elevated due to fructose treatment in SHR. Both fructose and DDW increased insulin level in WKY. Fructose did not affect NOS activity either in WKY or SHR. DDW increased NOS activity in LV of both WKY and SHR, while it decreased NOS activity and iNOS expression in the aorta of SHR with or without fructose treatment. In conclusion, DDW treatment significantly modified biochemical parameters in WKY together with NOS activity elevation in the heart. On the other hand, it did not affect biochemical parameters in SHR, but decreased NOS activity elevated due to iNOS upregulation in the aorta.

• MeSH
• aplikace orální MeSH
• deuterium izolace a purifikace   MeSH
• fruktosa aplikace a dávkování   MeSH
• hypertenze patofyziologie   MeSH
• konzumní sacharóza aplikace a dávkování   MeSH
• krevní tlak účinky léků   MeSH
• krysa rodu rattus MeSH
• oxid dusnatý metabolismus   MeSH
• potkani inbrední SHR MeSH
• srdce účinky léků   patofyziologie   MeSH
• voda aplikace a dávkování   chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The proposed project is based on recent findings documenting the imporatnce of insulin resistance syndrome in the pathogenesis of type 2 diabetes and cardiovascular diseases. The underlying pathophysiological mechanisms are not completely understood. Inan experimental model of insulin resistance, in hereditary hypertriglyceridemic rat strain and in the transgenic SHR rats overexpressing a SREBP-1 and resistin, the role and metabolic consequences of SREBP-1 and resistin will be analysed. The parametersto be evaluated include skeletal muscle and adipose tissue sensitivity to insulin action, lipid synthesis, transport and tissue accumulation, fatty acid composition of membrane phospholipids and activation of PPAR. The potential for nutritional (high sucrose diet, polyunsaturated fatty acids) and pharmacological intervention (fibrates, thiazolidinediones) will be evaluated.

Syndrom insulinové resistence je častou metabolickou poruchou výrazně zvyšující riziko rozvoje diabetu 2. typu a kardiovaskulárních onemocnění. Patoysiologické mechanismy nejsou dosud objasněny. U experimentálních modelů insulinové resistence, u neobesnílinie hereditárně hypertriglyceridemických potkanů a u transgenních spontánně hypertensních potkanů se zvýšenou expresí resistinu nebo SREBP-1 bude analysová úloha resistinu a SREBP-1. Sledována bude sensitivita tukové a svalové tkáně k účinku insulinu,substrátová kompetice mezi utilizací glukosy a NEMK ve tkáních, produkce a transport lipidů, spektrum mastných kyselin v membránových fosfolipidech a aktivace PPAR-alfa a -gama. Dále bude sledován vliv nutriční a farmakologické intervence na produkci aúčinky resistinu a aktivitu transkripčního faktoru SREBP-1.

• MeSH
• diabetes mellitus 2. typu MeSH
• hypertriglyceridemie MeSH
• inzulinová rezistence MeSH
• syntázy mastných kyselin MeSH
• thiazolidindiony MeSH

• Konspekt
• Fyziologie člověka a srovnávací fyziologie
• NLK Obory
• endokrinologie
• diabetologie
• vnitřní lékařství
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-D-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.

• MeSH
• Escherichia coli genetika   MeSH
• glukosidy metabolismus   MeSH
• glukosyltransferasy genetika   metabolismus   MeSH
• glykosylace MeSH
• rekombinantní proteiny MeSH
• rýže (rod) účinky léků   enzymologie   metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• trichotheceny metabolismus   toxicita   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MAIN CONCLUSION: We present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles. Chromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTrackerTM green, while their intactness and membrane potential were confirmed by staining with MitoTrackerTM orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae.

• MeSH
• Alveolata ultrastruktura   MeSH
• mikrořasy ultrastruktura   MeSH
• mitochondrie ultrastruktura   MeSH
• plastidy ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH