Flaxseed is an excellent source of valuable nutrients and is also considered a functional food. There are two types of hydrocolloids in flaxseed: flaxseed gum and proteins. Flaxseed gum exhibits emulsifying and foaming activities or can be used as a thickening and gelling agent. Due to its form of soluble fiber, flaxseed gum is related to many health benefits. Flaxseed proteins have various functional properties based on their physicochemical properties. While albumins possess the emulsion-forming ability, globulins better serve as foaming agents. Flaxseed proteins may also serve as a source of functional peptides with interesting biological and health-related activities. Functional properties and health-related benefits predetermine the application of these hydrocolloids, mainly in the food industry or medicine. Although these properties of flaxseed hydrocolloids have been recently and extensively studied, they are still not widely used on the industrial scale compared to other popular plant gums and proteins. The aim of this review was to present, discuss and highlight the recent discoveries in the structural characteristics and functional and biological properties of these versatile hydrocolloids with respect to factors affecting their characteristics and offer new insights into their potential applications as comparable alternatives to the other natural hydrocolloids or as the sources of novel functional products.

• Klíčová slova
• Linum usitatissimum L., flaxseed, flaxseed gum, flaxseed proteins, food hydrocolloids, functional properties, health benefits,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Genetic testing for cancer predisposition leads to the identification of a number of variants with uncertain significance. To some extent, variants of BRCA1/2 have been classified, in contrast to variants of other genes. CHEK2 is a typical example, in which a large number of variants of unknown clinical significance were identified and still remained unclassified. Herein, the CHEK2 variant assessment was performed through an in vivo, yeast-based, functional assay. In total, 120 germline CHEK2 missense variants, distributed along the protein sequence, and two large in-frame deletions were tested, originating from genetic test results in breast cancer families, or selected from the ClinVar database. Of these, 32 missense and two in-frame deletions behaved as non-functional, 73 as functional, and 15 as semi-functional, after comparing growth rates of each strain with positive and negative controls. The majority of non-functional variants were localized in the CHK2 kinase and forkhead-associated domains. In vivo results from the non-functional variants were in agreement with in silico predictions, and, where available, with strong breast cancer family history, to a great extent. The results of the largest, to date, yeast-based assay, evaluating CHEK2 variants, can complement and assist in the classification of rare CHEK2 variants with unclear clinical significance.

• Klíčová slova
• CHEK2 variants, breast cancer, functional assay, yeast,
• MeSH
• alely MeSH
• checkpoint kinasa 2 genetika   metabolismus   MeSH
• frekvence genu MeSH
• genetická predispozice k nemoci MeSH
• genetické asociační studie MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace * MeSH
• rodokmen MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• checkpoint kinasa 2 MeSH
• CHEK2 protein, human MeSH Prohlížeč

Isolated human hepatocytes (HH) are an accepted model for in vitro experiments for testing liver function and xenobiotic metabolism. Preferred over more traditional animal hepatocyte model used in toxicological studies, it is the model of choice when substances undergoing biotransformation in man are investigated. The aim of this study was to optimize isolation and culture conditions for HH primary culture with regard to cell yield, viability, and metabolic activity, and to evaluate the suitability of donor samples for toxicology experiments. Cell viability, total cytochrome P450 (CYP) content, CYP3A4, CYP1A2 activity, and finally mixed ethoxycoumarin-O-deethylase (ECOD) activity were parameters measured in order to characterize the isolated HH. The quality of the primary cultures, stable and functional for a seven-day period following 24 hour stabilization, was assessed by lactate dehydrogenase (LDH) leakage and response to the model toxin tert-butylhydroperoxide (tBH) and to silybinin, a model cytoprotective substance. Based on HH obtained from livers of five multiorgan donors (average age 44.8 years, three males and two females), the individual variability of donors needs to be considered in evaluating cultures focussing on clinical liver tests. Greater sensitivity to toxins and silybinin was found in the hepatocyte culture from one donor with higher aminotransferase activity. In another case, higher serum bilirubin appeared to be linked to higher ECOD activity. Our conclusion is that values of clinical liver tests ought to suggest a healthy organ thus eliminating previous hepatocyte damage, the crucial factor of primary culture stability and functioning.

• MeSH
• 7-alkoxykumarin-O-dealkylasa metabolismus   MeSH
• antioxidancia toxicita   MeSH
• blokátory kalciových kanálů toxicita   MeSH
• buněčné kultury metody   MeSH
• časové faktory MeSH
• cytochrom P-450 CYP1A2 metabolismus   MeSH
• cytochrom P-450 CYP3A MeSH
• dospělí MeSH
• hepatocyty chemie   účinky léků   fyziologie   MeSH
• inhibitory enzymů toxicita   MeSH
• jaterní mikrozomy účinky léků   MeSH
• játra účinky léků   metabolismus   MeSH
• kultivované buňky MeSH
• L-laktátdehydrogenasa metabolismus   toxicita   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nifedipin toxicita   MeSH
• oxygenasy se smíšenou funkcí metabolismus   MeSH
• rifampin toxicita   MeSH
• silymarin toxicita   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• terc-butylhydroperoxid toxicita   MeSH
• testy toxicity metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 7-alkoxykumarin-O-dealkylasa MeSH
• antioxidancia MeSH
• blokátory kalciových kanálů MeSH
• CYP3A protein, human MeSH Prohlížeč
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A2 MeSH
• cytochrom P-450 CYP3A MeSH
• inhibitory enzymů MeSH
• L-laktátdehydrogenasa MeSH
• nifedipin MeSH
• oxygenasy se smíšenou funkcí MeSH
• rifampin MeSH
• silymarin MeSH
• systém (enzymů) cytochromů P-450 MeSH
• terc-butylhydroperoxid MeSH

Large insert genomic DNA libraries are useful resources for genomic studies. Although the genome of Xenopus tropicalis stands as the amphibian reference genome because it benefitted from large-scale sequencing studies, physical mapping resources such as BAC libraries are lagging behind. Here we present the construction and characterization of a BAC library that covers the whole X. tropicalis genome. We prepared this BAC library from the genomic DNA of X. tropicalis females of the Adiopodoume strain. We characterized BAC clones by screening for specific loci, by chromosomal localization using FISH and by systematic BAC end sequencing. The median insert size is about 110kbp and the library coverage is around six genome equivalents. We obtained a total of 163,787 BAC end sequences with mate pairs for 77,711 BAC clones. We mapped all BAC end sequences to the reference X. tropicalis genome assembly to enable the identification of BAC clones covering specific loci. Overall, this BAC library resource complements the knowledge of the X. tropicalis genome and should further promote its use as a reference genome for developmental biology studies and amphibian comparative genomics.

• Klíčová slova
• Amphibian, BAC library, Genomics, Xenopus tropicalis,
• MeSH
• genomika metody   MeSH
• genová knihovna * MeSH
• hybridizace in situ fluorescenční MeSH
• játra chemie   MeSH
• mapování chromozomů MeSH
• proteiny Xenopus genetika   MeSH
• sekvenční analýza DNA MeSH
• umělé bakteriální chromozomy genetika   MeSH
• Xenopus genetika   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny Xenopus MeSH

Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.

• Klíčová slova
• Activity assay, Cathepsin B, Crystal structure, Expression, Inhibition, Parasite, Protease, Schistosoma mansoni,
• MeSH
• aktivace enzymů MeSH
• elektroporace MeSH
• exprese genu MeSH
• genetické vektory metabolismus   MeSH
• glykosylace MeSH
• kathepsin B antagonisté a inhibitory   chemie   izolace a purifikace   metabolismus   MeSH
• kinetika MeSH
• krystalizace MeSH
• mutace genetika   MeSH
• rekombinantní proteiny izolace a purifikace   metabolismus   MeSH
• Saccharomycetales genetika   MeSH
• Schistosoma mansoni enzymologie   MeSH
• transformace genetická MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kathepsin B MeSH
• rekombinantní proteiny MeSH

SMG7 proteins are evolutionary conserved across eukaryotes and primarily known for their function in nonsense mediated RNA decay (NMD). In contrast to other NMD factors, SMG7 proteins underwent independent expansions during evolution indicating their propensity to adopt novel functions. Here we characterized SMG7 and SMG7-like (SMG7L) paralogs in Arabidopsis thaliana. SMG7 retained its role in NMD and additionally appears to have acquired another function in meiosis. We inactivated SMG7 by CRISPR/Cas9 mutagenesis and showed that, in contrast to our previous report, SMG7 is not an essential gene in Arabidopsis. Furthermore, our data indicate that the N-terminal phosphoserine-binding domain is required for both NMD and meiosis. Phenotypic analysis of SMG7 and SMG7L double mutants did not indicate any functional redundancy between the two genes, suggesting neofunctionalization of SMG7L. Finally, protein sequence comparison together with a phenotyping of T-DNA insertion mutants identified several conserved regions specific for SMG7 that may underlie its role in NMD and meiosis. This information provides a framework for deciphering the non-canonical functions of SMG7-family proteins.

• Klíčová slova
• Arabidopsis, SMG7, gene duplication, meiosis, nonsense mediated RNA decay,
• Publikační typ
• časopisecké články MeSH

An increasing number of products containing synthetic cannabinoids pose a growing crisis to public health worldwide. Recently, a rising number of cases of serious adverse health effects, intoxications, and death cases associated with synthetic cannabinoids were reported. The current study represents the comprehensive structural analysis of three new synthetic cannabinoids (AB-, ADB- and AMB-FUBINACA) in solution investigated by electronic and vibrational circular dichroism together with the conventional methods of infrared and ultraviolet absorption spectroscopy, all supported by the density functional theory (DFT) calculations. The best level of theory to reproduce the experimental wavenumbers and wavelengths was found to be the B3PW91 method with a 6-311++G(d,p) basis set including the implicit solvent effect simulation. Very good agreement between the experimental and simulated spectra allowed us to determine the absolute configuration and a detailed interpretation of the IR absorption, VCD, ECD and UV spectra of AB-, ADB- and AMB-FUBINACA. In addition, the HOMO and LUMO electronic transitions were calculated.

• Klíčová slova
• Chiroptical spectroscopy, DFT calculation, Drugs, Synthetic cannabinoids, Vibrational spectroscopy,
• MeSH
• cirkulární dichroismus MeSH
• kanabinoidy * MeSH
• teorie funkcionálu hustoty MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kanabinoidy * MeSH

Tardigrades are microscopic ecdysozoans that can withstand extreme environmental conditions. Several tardigrade species undergo reversible morphological transformations and enter into cryptobiosis, which helps them to survive periods of unfavorable environmental conditions. However, the underlying molecular mechanisms of cryptobiosis are mostly unknown. Tubulins are evolutionarily conserved components of the microtubule cytoskeleton that are crucial in many cellular processes. We hypothesize that microtubules are necessary for the morphological changes associated with successful cryptobiosis. The molecular composition of the microtubule cytoskeleton in tardigrades is unknown. Therefore, we analyzed and characterized tardigrade tubulins and identified 79 tardigrade tubulin sequences in eight taxa. We found three α-, seven β-, one γ-, and one ε-tubulin isoform. To verify in silico identified tardigrade tubulins, we also isolated and sequenced nine out of ten predicted Hypsibius exemplaris tubulins. All tardigrade tubulins were localized as expected when overexpressed in mammalian cultured cells: to the microtubules or to the centrosomes. The presence of a functional ε-tubulin, clearly localized to centrioles, is attractive from a phylogenetic point of view. Although the phylogenetically close Nematoda lost their δ- and ε-tubulins, some groups of Arthropoda still possess them. Thus, our data support the current placement of tardigrades into the Panarthropoda clade.

• MeSH
• fylogeneze * MeSH
• Tardigrada * klasifikace   MeSH
• tubulin genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• tubulin MeSH

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

• Klíčová slova
• NAALADase, antibody fragment, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer, prostate-specific membrane antigen,
• MeSH
• antigeny povrchové imunologie   MeSH
• buněčné linie MeSH
• buňky PC-3 MeSH
• fluorescence MeSH
• glutamátkarboxypeptidasa II imunologie   MeSH
• hmyz MeSH
• jednořetězcové protilátky imunologie   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty imunologie   MeSH
• rekombinantní proteiny imunologie   MeSH
• xenogenní modely - testy protinádorové aktivity metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• jednořetězcové protilátky MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny MeSH

Claviceps paspali is used in the pharmaceutical industry for the production of ergot alkaloids. This fungus also biosynthesizes paspalitrems, indole diterpene (IDT) mycotoxins that cause significant economic losses in agriculture and represent safety concerns for ergot alkaloid manufacture. Here, we use Agrobacterium-mediated transformation to replace the idtP and the idtF genes in the IDT biosynthetic gene cluster of C. paspali with a selectable marker gene. We show that the ΔidtP knockout mutant produces paspaline, the first IDT intermediate of the pathway. The ΔidtF strain produces unprenylated IDTs such as paspalinine and paspaline. These experiments validate the function of idtP as the gene encoding the cytochrome P450 monooxygenase that oxidizes and demethylates paspaline to produce 13-desoxypaxilline, and that of idtF as the gene that encodes the α-prenyltransferase that prenylates paspalinine at the C20 or the C21 positions to yield paspalitrems A and C, respectively. In addition, we also show that axenic cultures of the wild type, the ΔidtP and the ΔidtF mutant C. paspali strains fail to produce an assembly of IDTs that are present in C. paspali-Paspalum spp. associations.

• MeSH
• biosyntetické dráhy genetika   MeSH
• Claviceps enzymologie   genetika   MeSH
• diterpeny metabolismus   MeSH
• geny hub * MeSH
• indoly metabolismus   MeSH
• multigenová rodina * MeSH
• oxygenasy se smíšenou funkcí genetika   MeSH
• prenyltransferáza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• diterpeny MeSH
• indoly MeSH
• oxygenasy se smíšenou funkcí MeSH
• prenyltransferáza MeSH