Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.

• MeSH
• autofagie účinky léků   genetika   MeSH
• benzylaminy farmakologie   MeSH
• chinazoliny farmakologie   MeSH
• endoplazmatické retikulum účinky léků   ultrastruktura   virologie   MeSH
• lidé MeSH
• mikrotubuly účinky léků   ultrastruktura   virologie   MeSH
• nádorové buněčné linie MeSH
• neurony účinky léků   ultrastruktura   virologie   MeSH
• nokodazol farmakologie   MeSH
• primární buněčná kultura MeSH
• replikace viru účinky léků   MeSH
• sirolimus farmakologie   MeSH
• tomografie elektronová MeSH
• virion účinky léků   růst a vývoj   ultrastruktura   MeSH
• viry klíšťové encefalitidy účinky léků   růst a vývoj   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

• MeSH
• analýza buněčné migrace metody   MeSH
• analýza hlavních komponent MeSH
• automatizace MeSH
• endozomy metabolismus   MeSH
• fenotyp MeSH
• fluorescenční mikroskopie MeSH
• lidé MeSH
• multivariační analýza MeSH
• nádorové buněčné linie MeSH
• nokodazol chemie   MeSH
• počítačové zpracování obrazu MeSH
• pohyb MeSH
• reprodukovatelnost výsledků MeSH
• RNA interference MeSH
• techniky kombinatorické chemie MeSH
• zelené fluorescenční proteiny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

• MeSH
• aktiny analýza   MeSH
• bicyklické sloučeniny heterocyklické farmakologie   MeSH
• buněčné jádro ultrastruktura   MeSH
• Cryptococcus neoformans účinky léků   fyziologie   ultrastruktura   MeSH
• dimethylsulfoxid farmakologie   MeSH
• elektronová mikroskopie MeSH
• faloidin analogy a deriváty   MeSH
• fluorescenční mikroskopie MeSH
• mikrofilamenta ultrastruktura   MeSH
• mikrotubuly účinky léků   MeSH
• modulátory tubulinu farmakologie   MeSH
• mořské toxiny farmakologie   MeSH
• nokodazol farmakologie   MeSH
• rhodaminy MeSH
• scavengery volných radikálů farmakologie   MeSH
• thiazolidiny farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.

• MeSH
• biologické modely MeSH
• CHO buňky MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• imunoblotting MeSH
• ionizující záření MeSH
• křečci praví MeSH
• lidé MeSH
• mitóza * fyziologie   účinky léků   MeSH
• molekulární sekvence - údaje MeSH
• nokodazol farmakologie   metabolismus   MeSH
• proteinkinasa CDC2 * metabolismus   MeSH
• proteinkinasy genetika   chemie   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• serin genetika   metabolismus   MeSH
• transfekce MeSH
• ultrafialové záření MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH

Microtubule disruptors, widely known as antimitotics, have broad applications in human medicine, especially as anti-neoplastic agents. They are subject to biotransformation within human body frequently involving cytochromes P450. Therefore antimitotics are potential culprits of drug-drug interactions on the level of activity as well as expression of cytochromes P450. This review discusses the effects of four well-known natural antimitotics: colchicine, taxol (paclitaxel), vincristine, and vinblastine, and a synthetic microtubule disruptor nocodazole on transcriptional activity of glucocorticoid and aryl hydrocarbon receptors. It appears that microtubules disarray restricts the signaling by these two nuclear receptors regardless of cell cycle phase. Consequently, intact microtubules play an important role in the regulation of expression of cytochromes P450, which are under direct or indirect control of the two nuclear receptors.

• MeSH
• antimitotika farmakologie   MeSH
• biotransformace MeSH
• kolchicin farmakologie   MeSH
• lékové interakce MeSH
• lidé MeSH
• mikrotubuly účinky léků   MeSH
• nokodazol farmakologie   MeSH
• paclitaxel farmakologie   MeSH
• receptory aromatických uhlovodíků metabolismus   MeSH
• receptory glukokortikoidů metabolismus   MeSH
• signální transdukce MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• vinblastin farmakologie   MeSH
• vinkristin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• HeLa buňky účinky léků   MeSH
• kolchicin aplikace a dávkování   chemie   MeSH
• nokodazol aplikace a dávkování   chemie   MeSH
• proteiny asociované s mikrotubuly MeSH
• receptory glukokortikoidů fyziologie   ultrastruktura   MeSH
• vinkristin MeSH
• Publikační typ
• kongresy MeSH

In this study we examined whether microtubules and heat shock protein 90 (Hsp90) are involved in phorbol myristate acetate (PMA) and N-formyl-Met-Leu-Phe (fMLP)-induced oxidative burst in DMSO-differentiated HL-60 cells. Our results showed that microtubule interfering agents, paclitaxel (1-5 microM), colchicine (1-100 microM), nocodazole (1-20 microM), and vincristine (1-50 microM), did not affect either PMA or fMLP-induced oxidative burst. In contrast, radicicol, an inhibitor of Hsp90, inhibited fMLP-induced oxidative burst in time and concentration-dependent manner where IC50 value for 30 min pre-incubation was 16.5 +/- 3.5 microM radicicol. We conclude that both PMA and fMLP-induced oxidative burst in DMSO-differentiated HL-60 cells is microtubule-independent while the latter requires Hsp90 activity.

• MeSH
• buněčná diferenciace MeSH
• dimethylsulfoxid farmakologie   MeSH
• HL-60 buňky MeSH
• kolchicin farmakologie   MeSH
• lidé MeSH
• mikrotubuly účinky léků   fyziologie   MeSH
• N-formylmethionin-leucyl-fenylalanin farmakologie   MeSH
• neutrofily metabolismus   MeSH
• nokodazol farmakologie   MeSH
• paclitaxel farmakologie   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• respirační vzplanutí účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH