We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient. An enriched lysosomal membrane fraction was collected from the 20%/35% sucrose interface. The washed lysosomal membrane fraction was enriched 30 times relative to the homogenate and gave the yield of more than 8%. These results are comparable to lysosomal membranes isolated by magnetic chromatography from cultured cells (Diettrich et al., 1998). The procedure effectively eliminated mitochondrial contamination and minimized contamination from other cell compartments. The enriched fractions retained the ability to acidify membrane vesicles through the activity of lysosomal vacuolar ATPase. The method avoids non-physiological overloading of cells with superparamagnetic particles and appears to be quite robust among the tested cell lines. We expect it may be of more general use, adaptable to other cell lines and tissues.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• centrifugace - gradient hustoty MeSH
• frakcionace buněk metody   MeSH
• glukosylceramidasa metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• intracelulární membrány účinky léků   metabolismus   MeSH
• kyseliny metabolismus   MeSH
• lidé MeSH
• lyzozomy účinky léků   metabolismus   MeSH
• subcelulární frakce účinky léků   metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lysosomální onemocnění jsou vzácné, dědičně podmíněné nemoci způsobené nedostatečnou aktivitou některého z lysosomálních enzymů, event. transportních proteinů. První příznaky se mohou objevit kdykoli od novorozeneckého období až do pozdní dospělosti, časné formy mívají těžký průběh s rychlou progresí a infaustní prognózou. Postižení je multisystémové s trvalou progresí obtíží a postižením metabolicky aktivních orgánů či tkání – kostní dřeň, játra, kosti, kosterní svaly, myokard, CNS. Diagnózu definitivně potvrdíme průkazem snížené aktivity daného enzymu a mutační analýzou. Některé ze střádavých nemocí můžeme účinně léčit podáním rekombinantních enzymů intravenózně či omezením množství střádaného substrátu. U malého počtu lysosomálních onemocnění je úspěšná transplantace kostní dřeně. Je nutná mezioborová spolupráce včetně zajištění genetického poradenství a prenatální diagnostiky v rodinách pacientů. První část sdělení je věnována obecné charakteristice lysosomálních onemocnění a problematice nejčastějších onemocnění, které je možné v současné době v České republice léčit (Gaucherova nemoc, Pompeho nemoc, Fabryho nemoc, Niemann-Pickova nemoc, nemoc ze střádání esteru cholesterolu). Ve druhé části článku je věnována pozornost problematice mukopolysacharidóz, které představují další skupinu vzácných lysosomálních onemocnění.

Lysosomal storage diseases are rare genetic diseases caused by insufficient activity of some of the lysosomal enzymes and/or transport proteins. Initial symptoms may appear any time from the neonatal period to late adulthood; early forms tend to have a severe course with rapid progression and unfavorable prognosis. There is multisystem involvement with continuous progression of symptoms and involvement of metabolically active organs or tissues – the bone marrow, liver, bones, skeletal muscles, myocardium, or CNS. The diagnosis is definitively confirmed by demonstration of reduced activity of the particular enzyme and by mutation analysis. Some of the storage diseases can be effectively treated by intravenous administration of recombinant enzymes or by limiting the amount of the substrate stored. In a small number of lysosomal storage diseases, bone marrow transplantation is successful. Multidisciplinary collaboration, including genetic counseling and prenatal diagnosis in patient families, is required. The first part of the paper deals with general characteristics of lysosomal storage diseases and the most common diseases that are currently treatable in the Czech Republic (Gaucher’s disease, Pompe disease, Fabry disease, Niemann–Pick disease, cholesterol ester storage disease). The second part of the paper deals with mucopolysaccharidoses, another group of rare lysosomal storage diseases.

• Klíčová slova
• enzymová substituční léčba, substrát redukční terapie,
• MeSH
• 1-deoxynojirimycin analogy a deriváty   MeSH
• alfa-glukosidasy MeSH
• časná diagnóza MeSH
• dítě MeSH
• dospělí MeSH
• enzymová substituční terapie metody   využití   MeSH
• Fabryho nemoc diagnóza   farmakoterapie   patofyziologie   MeSH
• Gaucherova nemoc diagnóza   farmakoterapie   klasifikace   MeSH
• genetické nemoci vrozené * diagnóza   farmakoterapie   klasifikace   MeSH
• genistein MeSH
• glykogenóza typu II diagnóza   farmakoterapie   klasifikace   MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory glykosidových hydrolas MeSH
• inhibitory proteinkinas MeSH
• kojenec MeSH
• lidé MeSH
• lyzozomální nemoci z ukládání * diagnóza   ekonomika   farmakoterapie   klasifikace   MeSH
• mladiství MeSH
• mukopolysacharidózy diagnóza   ekonomika   terapie   MeSH
• Niemannova-Pickova nemoc typu C diagnóza   farmakoterapie   patofyziologie   MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• příznaky a symptomy MeSH
• vzácné nemoci * diagnóza   farmakoterapie   klasifikace   MeSH
• Wolmanova nemoc MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

The sheep tapeworm Moniezia expansa is very common parasite, which affects ruminants such as sheep, goats as well as other species. The benzimidazole anthelmintics albendazole (ABZ), flubendazole (FLU) and mebendazole (MBZ) are often used to treat the infection. The drug-metabolizing enzymes of helminths may alter the potency of anthelmintic treatment. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (ABZ, MBZ and FLU) in M. expansa. Activities of biotransformation enzymes were determined in subcellular fractions. Metabolites of the anthelmintics were detected and identified using high performance liquid chromatography/ultra-violet/VIS/fluorescence or ultra-high performance liquid chromatography/mass spectrometry. Reduction of MBZ, FLU and oxidation of ABZ were proved as well as activities of various metabolizing enzymes. Despite the fact that the conjugation enzymes glutathione S-transferase, UDP-glucuronosyl transferase and UDP-glucosyl transferase were active in vitro, no conjugated metabolites of anthelmintics were identified either ex vivo or in vitro. The obtained results indicate that sheep tapeworm is able to deactivate the administered anthelmintics, and thus protects itself against their action.

• MeSH
• albendazol farmakokinetika   farmakologie   MeSH
• alkoholoxidoreduktasy metabolismus   MeSH
• anthelmintika farmakokinetika   farmakologie   MeSH
• biotransformace MeSH
• Cestoda účinky léků   enzymologie   ultrastruktura   MeSH
• glukuronosyltransferasa metabolismus   MeSH
• glutathiontransferasa metabolismus   MeSH
• izoenzymy metabolismus   MeSH
• katalasa metabolismus   MeSH
• mebendazol analogy a deriváty   farmakokinetika   farmakologie   MeSH
• monieziáza parazitologie   MeSH
• multienzymové komplexy metabolismus   MeSH
• NADH, NADPH oxidoreduktasy metabolismus   MeSH
• nemoci ovcí parazitologie   MeSH
• ovce MeSH
• oxidace-redukce MeSH
• oxygenasy se smíšenou funkcí metabolismus   MeSH
• peroxidasa metabolismus   MeSH
• superoxiddismutasa metabolismus   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• tenké střevo parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.

• MeSH
• biologický transport MeSH
• kultivované buňky MeSH
• kyselina 2,4-dichlorfenoxyoctová chemie   metabolismus   MeSH
• kyseliny indoloctové chemie   metabolismus   MeSH
• naftaleny chemie   metabolismus   MeSH
• regulátory růstu rostlin chemie   metabolismus   MeSH
• tabák chemie   cytologie   metabolismus   MeSH
• teoretické modely MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AND AIMS: The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [(3)H]trans-zeatin (transZ) and auxin ([(3)H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. METHODS: Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [(3)H]transZ and [(3)H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. KEY RESULTS: The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [(3)H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division. CONCLUSIONS: Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

• MeSH
• Chlorophyta chemie   metabolismus   fyziologie   MeSH
• cytokininy analýza   metabolismus   MeSH
• fylogeneze MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• homeostáza fyziologie   MeSH
• kyseliny indoloctové analýza   metabolismus   MeSH
• regulátory růstu rostlin analýza   metabolismus   MeSH
• sinice chemie   metabolismus   fyziologie   MeSH
• Streptophyta chemie   metabolismus   fyziologie   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH