Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

• Klíčová slova
• African trypanosomiasis, Trypanosoma brucei, microtubules, photoaffinity labeling, seventh site, triazolopyrimidines, vinca site,
• MeSH
• HEK293 buňky MeSH
• lidé MeSH
• proteomika MeSH
• pyrimidiny chemie   MeSH
• savci metabolismus   MeSH
• trypanocidální látky * chemie   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• trypanozomóza africká * farmakoterapie   MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• pyrimidiny MeSH
• trypanocidální látky * MeSH
• tubulin MeSH

Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available. In this review, the basic techniques used for cell quantification, with a special emphasis on techniques based on fluorescent DNA dyes, are described. The main aim of this review is to guide readers through the possibilities of cell quantification with various methods and to show the strengths and weaknesses of these methods, especially with respect to their sensitivity, accuracy, and length. As these methods are frequently accompanied by an analysis of cell proliferation and cell viability, some of these approaches are also described.

• Klíčová slova
• DNA dyes, cell metabolism, cell quantification, enzymatic conversion of substrate,
• MeSH
• biotest MeSH
• DNA analýza   chemie   metabolismus   MeSH
• fluorescenční barviva * MeSH
• počet buněk metody   MeSH
• proliferace buněk MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA MeSH
• fluorescenční barviva * MeSH

The microbial biofilms are ubiquitous in nature and represent important biological entities that affect various aspects of human life. As such, they attracted considerable attention during last decades, with the factors affecting the biofilm development being among the frequently studied topics. In our work, the biofilm was cultivated on the surface of polypropylene fibers in a nutrient medium inoculated by the suspension of two unsterile soils. The effects of ionic strength and valence of salt on the amount of the produced biofilm and on composition of biofilm microbial communities were investigated. The effect of valence was significant in some OTUs: Arthrobacter/Pseudarthrobacter/Paenarthrobacter and Bacillus with positive response to monovalent salt (KCl) and Streptomyces, Lysinibacillus, Pseudomonas, and Ensifer with positive response to divalent salt (MgSO4). The significant preference for a certain concentration of salts was observed in the case of OTUs Agrobacterium, Bacillus (both 100 mM), and Brevundimonas (30 mM). A new quantification method based on measuring of oxidizable organic carbon in biofilm biomass, based on dichromate oxidation, was used. We compared the results obtained using this method with results of crystal violet destaining and measuring of extracted DNA concentration as proxies of the biofilm biomass. The dichromate oxidation is simple, inexpensive, and fast, and our results show that it may be more sensitive than crystal violet destaining. The highest biomass values tended to associate with high concentrations of the divalent salt. This trend was not observed in treatments where the monovalent salt was added. Our data confirm the importance of inorganic ions for biofilm composition and biomass accumulation.

• Klíčová slova
• Biofilm, Dichromate oxidation, Ionic strength, Quantification, Valence,
• MeSH
• Bacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• bakteriologické techniky MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• biomasa MeSH
• kultivační média chemie   MeSH
• mikrobiota účinky léků   MeSH
• minerály analýza   farmakologie   MeSH
• polypropyleny MeSH
• půdní mikrobiologie MeSH
• soli analýza   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kultivační média MeSH
• minerály MeSH
• polypropyleny MeSH
• soli MeSH

Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.

• MeSH
• barvení a značení metody   MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• cytofotometrie metody   MeSH
• diploidie * MeSH
• DNA analýza   chemie   MeSH
• dodecylsíran sodný chemie   MeSH
• fluorescenční barviva chemie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• počet buněk přístrojové vybavení   metody   MeSH
• replikace DNA * MeSH
• reprodukovatelnost výsledků MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• dodecylsíran sodný MeSH
• fluorescenční barviva MeSH

The relative potencies of non-ortho-substituted coplanar polychlorinated biphenyl (PCB) congeners to activate the aryl hydrocarbon receptor (AhR) and to cause the AhR-dependent toxic events are essential for their risk assessment. Since some studies suggested that abundant non-dioxin-like PCB congeners (NDL-PCBs) may alter the AhR activation by PCB mixtures and possibly cause non-additive effects, we evaluated potential suppressive effects of NDL-PCBs on AhR activation, using a series of 24 highly purified NDL-PCBs. We investigated their impact on the model AhR agonist-induced luciferase reporter gene expression in rat hepatoma cells and on induction of CYP1A1/1B1 mRNAs and deregulation of AhR-dependent cell proliferation in rat liver epithelial cells. PCBs 128, 138, and 170 significantly suppressed AhR activation (with IC50 values from 1.4 to 5.6 μM), followed by PCBs 28, 47, 52, and 180; additionally, PCBs 122, 153, and 168 showed low but still significant potency to reduce luciferase activity. Detection of CYP1A1 mRNA levels in liver epithelial cells largely confirmed these results for the most abundant NDL-PCBs, whereas the other AhR-dependent events (CYP1B1 mRNA expression, induction of cell proliferation in confluent cells) were less sensitive to NDL-PCBs, thus indicating a more complex regulation of these endpoints. The present data suggest that some NDL-PCBs could modulate overall dioxin-like effects in complex mixtures.

• Klíčová slova
• Aryl hydrocarbon receptor, Cytochrome P450, DR-CALUX® assay, Disruption of contact inhibition, NDL-PCBs, Relative effect potency,
• MeSH
• buněčné linie MeSH
• cytochrom P-450 CYP1A1 genetika   metabolismus   MeSH
• epitelové buňky cytologie   účinky léků   metabolismus   MeSH
• exprese genu účinky léků   MeSH
• hepatocyty cytologie   účinky léků   metabolismus   MeSH
• játra účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• polychlorované bifenyly chemie   toxicita   MeSH
• proliferace buněk účinky léků   MeSH
• receptory aromatických uhlovodíků genetika   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytochrom P-450 CYP1A1 MeSH
• polychlorované bifenyly MeSH
• receptory aromatických uhlovodíků MeSH

The ability of C. parapsilosis (an important cause of nosocomial infections) to produce biofilm was evaluated in 32 bloodstream isolates and 85 strains isolated from skin. The biofilm formation was found in 19 (59%) blood isolates and only in 33 (39%) isolates from skin. The antifungal susceptibility was assessed for amphotericin B, itraconazole and voriconazole in planktonic and biofilm form of the 19 biofilm-positive bloodstream strains by broth microdilution method according to NCCLS standards. The method was modified by the use of resazurin as a colorimetric indicator of the metabolically active cells which makes the determination of the effect of antifungal agents easier. Biofilm forms of all strains were more resistant than their planktonic form.

• MeSH
• antifungální látky farmakologie   MeSH
• biofilmy účinky léků   MeSH
• Candida účinky léků   patogenita   MeSH
• fungální léková rezistence účinky léků   MeSH
• fungemie farmakoterapie   MeSH
• infekce spojené se zdravotní péčí mikrobiologie   MeSH
• kolorimetrie MeSH
• lidé MeSH
• mikrobiální testy citlivosti metody   MeSH
• mikrobiologické techniky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antifungální látky MeSH