Elektron kryo-mikroskopické techniky v biologickém výzkumu a nanotechnologiích

Příprava biologických vzorků pro transmisní elektronovou mikroskopii není triviální úkol. Vzorky musí odolat vakuu přítomném v mikroskopu, a proto je často nutné uplatnit nefyziologické postupy při jejich zpracování. Tyto postupy obvykle zahrnují fixaci na bázi aldehydů, nahrazení vody alkoholem (t.j. dehydrataci/substituci), a zalití do pryskyřice, která vytváří podporu pro následnou přípravu tenkých řezů, které pak mohou být vloženy do mikroskopu. V posledním desetiletí získala dominantní postavení v oblasti výzkumu buněčné biologie metoda kryo-fixace (vitrifikace) za pomoci ultrarychlého vysokotlakého zmrazování a následná kryo-substituce a zalití vzorků do pryskyřice při nízkých teplotách. Tímto způsobem byli úspěšně vitrifikovány různé biologické vzorky s tloušťkou až několik stovek mikrometrů do stavu, který byl srovnatelný s jejich in vivo strukturou. Kryo-fixace izolovaných biologických objektů (s omezenou tloušťkou do několika mikrometrů) je možná i v tenké vrstvě vitrifikované vody za pomoci imerzní kryo-fixace při normálním tlaku. V kombinaci s kryo-elektronovou mikroskopií se tato metoda stala nejefektivnejším a základním principem pro tvorbu elektron kryo-mikroskopických obrázků plně hydratovaných vzorků s velmi vysokým rozlišením na úrovni několika desetin nanometrů. Obě tyto metody...Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...Ústav buněčné biologie a patologie 1. LF UKInstitute of Cell Biology and Pathology First Faculty of Medicine Charles UniversityFirst Faculty of Medicine1. lékařská fakult

Pathogenetical Characterization of MHV-76: a Spontaneous 9.5-Kilobase-Deletion Mutant of Murine Lymphotropic Gammaherpesvirus 68

Murid gammaherpesvirus 4 (MuHV-4) provides a small animal model for the study of animal gammaherpesviruses. MHV-76 is a spontaneous deletion mutant as compared to the prototype strain of MuHV-4 (MHV-68). The MHV-76 genome lacks at least 12 ORFs at the 5'-end including the M1, M2, M3 and M4 genes and the eight viral t-RNA-like genes. During 27 months of experimental infection of BALB/c mice we followed their pathogenesis, immunology and oncogenic properties. After intranasal infection with MHV-76, the infectious virus was detected in the blood, thymus, lungs, heart, liver, spleen, bone marrow, peritoneal macrophages, lymph nodes, kidneys, mammary glands, brain and small intestine. The acute phase of infection was attenuated, but the chronic phase of infection was accompanied with long persistence of virus not only in the lymphatic, but in the neural and glandular tissue, as well. In comparison with the prototype strain, splenomegaly and lymphocytosis was very low. Surprisingly, during 27 months the BALB/c mice infected with MHV-76 did not develop lymphoproliferative disorders like infectious mononucleosis, leukaemia or lymphomas. We hypothesize that the M4 gene, present in all oncogenic MHV isolates, might be related (directly or indirectly) to their transforming properties

Static analysis of the cylindrical tank resting on various types of subsoil

The paper presents analyses of deformations and stress of the circular rotationally symmetric tanks. The reinforced concrete tanks – water reservoirs – resting on elastic subsoil have been analyzed. The elastic subsoil has been modeled using three basic subsoil models: half-space model, two-parametric model and one-parametric (Winkler) subsoil model. Particular modifications have been applied to the basic models which yield in seven models presented. The cylindrical tank has been analyzed in the interaction with the subsoil, while the elastic subsoil has been characterized by different parameters. The differences in modeling using individual calculation models have been highlighted. The results from analytical and numerical solutions have been presented and compared in graphs and tables. The method for the calculation of settlement of the circular foundation plate was verified experimentally. The settlement of the circular plate was measured while the silo was fully loaded by its contents. The experimentally obtained values have been compared with the calculation results. In the conclusion the recommendations for the suitable model subsoil for these types of structures have been presented

Comparative Analysis Reveals Changes in Some Seed Properties in Amaranth Mutant Variety ‘Zobor’ (A. hypochondriacus × A. hybridus)

The aim of our long-term research program is to improve the quality and quantity of amaranth production through mutation breeding using γ-radiation. In this paper, we present the characterization of the new variety ‘Zobor’ of A. hypochondriacus × A. hybridus developed by radiation-induced mutagenesis of hybrid K-433. Multiyear phenotypic characterization of an important yield parameter (1000-seed weight) showed that the studied mutant variety ‘Zobor’ has an advantage in seed weight over the nonirradiated control seeds of K-433 with predictable performance of this yield trait. ‘Zobor’ exhibited changes in seed morphometric parameters, starch particle size, and pasting properties with no change in amylose content and swelling power. Moreover, the seeds of ‘Zobor’ showed the significantly highest folate content among selected amaranth varieties. The mutant variety could, therefore, be interesting for the development of functional foods and as a low-management crop, attractive for cultivation in Europe

Comparative Analysis Reveals Changes in Some Seed Properties in Amaranth Mutant Variety ‘Zobor’ (A. hypochondriacus × A. hybridus)

The aim of our long-term research program is to improve the quality and quantity of amaranth production through mutation breeding using γ-radiation. In this paper, we present the characterization of the new variety ‘Zobor’ of A. hypochondriacus × A. hybridus developed by radiation-induced mutagenesis of hybrid K-433. Multiyear phenotypic characterization of an important yield parameter (1000-seed weight) showed that the studied mutant variety ‘Zobor’ has an advantage in seed weight over the nonirradiated control seeds of K-433 with predictable performance of this yield trait. ‘Zobor’ exhibited changes in seed morphometric parameters, starch particle size, and pasting properties with no change in amylose content and swelling power. Moreover, the seeds of ‘Zobor’ showed the significantly highest folate content among selected amaranth varieties. The mutant variety could, therefore, be interesting for the development of functional foods and as a low-management crop, attractive for cultivation in Europe

Study on metal-triggered callose deposition in roots of maize and soybean

Abstract: Callose plays important roles in a variety of processes of plant development, and/or in a response to a range of biotic and abiotic stresses. In the current work we have studied and compared the effect of lead, cadmium and arsenic on accumulation of newly formed callose deposits in the roots of maize and soybean. We observed formation of characteristic callose deposits in the root cell walls, probably associated with plasmodesmata, depending on the type of metal and the plant species investigated. Further, the callose turnover was analysed by measuring of total callose content as well as activities of total β-(1,3)-glucanases in roots. The latter enzymes are responsible for callose depletion, and their possible role during metal stress has previously been proposed. However, neither of these biochemical values appeared to be sufficiently reliable for scoring the altered callose turnover (including local deposits) in plant tissue. The microscopical observations are discussed in light of the biochemical data obtained

Cyclin-dependent kinase 8 represents a positive regulator of cytomegalovirus replication and a novel host target for antiviral strategies

Background. Cyclin-dependent kinase 8 (CDK8) is a multifaceted regulator and represents a catalytic component of the transcriptional Mediator complex. CDK8 activity, on the one hand, increases transcriptional elongation by the recruitment of Mediator/super elongation complexes, but, on the other hand, negatively regulates CDK7-controlled transcriptional initiation through inactivating cyclin H phosphorylation. Recently, these combined properties of CDK8 have also suggested its rate-limiting importance for herpesviral replication. Objectives. In this paper, we focused on human cytomegalovirus (HCMV) and addressed the question of whether the pharmacological inhibition or knock-down of CDK8 may affect viral replication efficiency in cell culture models. Methods. A number of human and animal herpesviruses, as well as non-herpesviruses, were used to analyze the importance of CDK8 for viral replication in cell culture models, and to assess the antiviral efficacy of CDK8 inhibitors. Results. Using clinically relevant CDK8 inhibitors (CCT-251921, MSC-2530818, and BI-1347), HCMV replication was found strongly reduced even at nanomolar drug concentrations. The EC50 values were consistent for three different HCMV strains (i.e., AD169, TB40, and Merlin) analyzed in two human cell types (i.e., primary fibroblasts and astrocytoma cells), and the drugs comprised a low level of cytotoxicity. The findings highlighted the following: (i) the pronounced in vitro SI values of anti-HCMV activity obtained with CDK8 inhibitors; (ii) a confirmation of the anti-HCMV efficacy by CDK8–siRNA knock-down; (iii) a CDK8-dependent reduction in viral immediate early, early, and late protein levels; (iv) a main importance of CDK8 for viral late-stage replication; (v) several mechanistic aspects, which point to a strong impact on viral progeny production and release, but a lack of CDK8 relevance for viral entry or nuclear egress; (vi) a significant anti-HCMV drug synergy for combinations of inhibitors against host CDK8 and the viral kinase vCDK/pUL97 (maribavir); (vii) finally, a broad-spectrum antiviral activity, as seen for the comparison of selected α-, β-, γ-, and non-herpesviruses. Conclusions. In summary, these novel data provide evidence for the importance of CDK8 as a positive regulator of herpesviral replication efficiency, and moreover, suggest its exploitability as an antiviral target for novel strategies of host-directed drug development

Derivates of 5-methylpyrazine-2-carboxylic Acid as Potential Drugs

Title of rigorous thesis: Derivates of 5-methylpyrazine-2-carboxylic acid as potential drugs Introduction of rigorous thesis is about tuberculosis, its occurence in the world, drug resistence, pathogenesis, diagnosis, prevention and treatment, about used and developed drugs. Further, I describe methods of carboxylic acid amides preparation, schemes of reactions and six prepared anilides. These are characterized by melting temperature, elemental analysis, IR, 1 H and 13 C NMR spectroscopy. Synthetized compounds were tested in vitro for antituberculotic, antifungal and herbicidal activity. Also values of lipophilicity log P and log K were gained. Relationship between biological activity, chemical structure and lipophilicity of prepared substances was evaluated