Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.

• MeSH
• artemisininy izolace a purifikace   metabolismus   MeSH
• axenická kultura MeSH
• berberin izolace a purifikace   metabolismus   MeSH
• biologické přípravky izolace a purifikace   metabolismus   MeSH
• biotechnologie metody   MeSH
• buněčné kultury MeSH
• fytonutrienty biosyntéza   izolace a purifikace   MeSH
• metabolické inženýrství metody   MeSH
• naftochinony izolace a purifikace   metabolismus   MeSH
• paclitaxel biosyntéza   izolace a purifikace   MeSH
• rostlinné buňky chemie   metabolismus   MeSH
• rostliny chemie   genetika   metabolismus   MeSH
• sekundární metabolismus MeSH
• techniky tkáňových kultur MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

To find an effective drug for Zika virus, it is important to understand how numerous proteins which are critical for the virus' structure and function interact with their counterparts. One approach to inhibiting the flavivirus is to deter its ability to bind onto glycoproteins; however, the crystal structures of envelope proteins of the ever-evolving viral strains that decipher glycosidic or drug-molecular interactions are not always available. To fill this gap, we are reporting a holistic, simulation-based approach to predict compounds that will inhibit ligand binding onto a structurally unresolved protein, in this case the Zika virus envelope protein (ZVEP), by developing a three-dimensional general structure and analyzing sites at which ligands and small drug-like molecules interact. By examining how glycan molecules and small-molecule probes interact with a freshly resolved ZVEP homology model, we report the susceptibility of ZVEP to inhibition via two small molecules, ZINC33683341 and ZINC49605556-by preferentially binding onto the primary receptor responsible for the virus' virulence. Antiviral activity was confirmed when ZINC33683341 was tested in cell culture. We anticipate the results to be a starting point for drug discovery targeting Zika virus and other emerging pathogens.

• MeSH
• antivirové látky chemie   farmakologie   MeSH
• Cercopithecus aethiops MeSH
• knihovny malých molekul chemie   farmakologie   MeSH
• molekulární modely MeSH
• počítačová simulace MeSH
• polysacharidy metabolismus   MeSH
• proteiny virového obalu antagonisté a inhibitory   chemie   MeSH
• strukturní homologie proteinů MeSH
• vazebná místa MeSH
• Vero buňky MeSH
• virová nálož účinky léků   MeSH
• virus zika účinky léků   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Celiac disease (CD) is an autoimmune enteropathy initiated and sustained by the ingestion of gluten in genetically susceptible individuals. It is caused by a dysregulated immune response toward both dietary antigens, the gluten proteins of wheat, rye, and barley, and autoantigens, the enzyme tissue transglutaminase (TG2). The small intestine is the target organ. Although routine immunochemical protocols for a laboratory diagnosis of CD are available, faster, easier-to-use, and cheaper analytical devices for CD diagnosis are currently unavailable. This review focuses on biosensors, consisting of a physicochemical transducer and a bioreceptor, as promising analytical tools for diagnosis of CD and other diseases. Examples of recently developed biosensors as well as expectations for future lines of research and development in this field are presented.

• MeSH
• biosenzitivní techniky metody   MeSH
• celiakie diagnóza   imunologie   MeSH
• elektrochemické techniky MeSH
• gliadin imunologie   MeSH
• imunohistochemie MeSH
• lidé MeSH
• proteiny vázající GTP imunologie   MeSH
• protilátky metabolismus   MeSH
• transglutaminasy imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Laccases are multi-copper oxidoreductases which catalyze the oxidation of a wide range of substrates during the simultaneous reduction of oxygen to water. These enzymes, originally found in fungi, plants, and other natural sources, have many industrial and biotechnological applications. They are used in the food, textile, pulp, and paper industries, as well as for bioremediation purposes. Although natural hosts can provide relatively high levels of active laccases after production optimization, heterologous expression can bring, moreover, engineered enzymes with desired properties, such as different substrate specificity or improved stability. Hence, diverse hosts suitable for laccase production are reviewed here, while the greatest emphasis is placed on yeasts which are commonly used for industrial production of various proteins. Different approaches to optimize the laccase expression and activity are also discussed in detail here.

• MeSH
• Bacteria genetika   MeSH
• hmyz cytologie   genetika   MeSH
• houby genetika   MeSH
• kvasinky genetika   MeSH
• lakasa genetika   metabolismus   MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• substrátová specifita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved.

• MeSH
• enzymy imobilizované metabolismus   MeSH
• glukosaoxidasa metabolismus   MeSH
• kolorimetrie metody   MeSH
• krevní glukóza metabolismus   MeSH
• lidé MeSH
• magnetismus * MeSH
• peroxidasy metabolismus   MeSH
• spektrofotometrie MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.

• MeSH
• aminohydrolasy chemie   genetika   metabolismus   MeSH
• Arthrodermataceae enzymologie   MeSH
• Aspergillus niger enzymologie   MeSH
• druhová specificita MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• koncentrace vodíkových iontů MeSH
• kyseliny mandlové metabolismus   MeSH
• Nectria enzymologie   MeSH
• Neurospora crassa enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

High-resolution melting curve analysis (HRM) of polymerase chain reaction (PCR) amplicons has been described as a fast, cheap, and reliable closed-tube method of genotyping with no need for labeled primers or labeled probes. We adapted this melting analysis assay for the detection of the most common nonfunctional alleles of cytochrome P-450 (CYP) 2D6 in the Caucasian population that affect the metabolism of many commonly used drugs. We used this method to genotype 91 patients under paroxetine therapy. The presence and the constitution of the most common single-nucleotide polymorphisms (1846G>A, 2988G>A, 100C>T, 2549delA, 2615_2617delAAG, and 1707delT) in poor and intermediate metabolizers from the Caucasian population were detected in short amplicons (≤148 bp). After fluorescence normalization, the wild-type, homozygous, and heterozygous samples were easily distinguishable from each other by their specific melting curve shape. A total of 92.6% of the 1846G>A heterozygotes, 96% of the 100C>T heterozygotes, and 100% of the 2988G>A, 2549delA, 2615_2617delAAG, and 1707delT heterozygotes have been correctly distinguished from the wild types. One hundred percent of all the homozygotes in this group of patients have been detected without any error. HRM of short amplicons is a simple tool for effective, rapid, and reliable CYP2D6 genotyping that does not require real-time PCR, labeled probes, processing or any separations after PCR. The reaction is performed in a closed-tube system and is highly specific and sensitive. We proved that this technique is highly reliable for use in routine diagnostics.

• MeSH
• alely MeSH
• běloši genetika   MeSH
• cytochrom P-450 CYP2D6 genetika   metabolismus   MeSH
• DNA primery genetika   MeSH
• frekvence genu MeSH
• genotypizační techniky metody   MeSH
• heterozygot MeSH
• homozygot MeSH
• jednonukleotidový polymorfismus MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Escherichia coli strains expressing different nitrilases transformed nitriles or KCN. Six nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetonitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising nitrilases for the transformation of industrially important substrates were found: the nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the nitrilases from N. crassa and A. niger for (R,S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine.

• MeSH
• aminohydrolasy chemie   genetika   metabolismus   MeSH
• dehydratasy chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• genom fungální * MeSH
• genomika MeSH
• houby enzymologie   genetika   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: -43, -44, -49, and -50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions -49 and -50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.

• MeSH
• bakteriální geny MeSH
• biomasa MeSH
• biotechnologie MeSH
• kyselina hyaluronová chemie   metabolismus   MeSH
• mutace * MeSH
• mutageneze cílená metody   MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) MeSH
• Streptococcus equi genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The choice of tools that enable efficient targeting of exogenous antigens (Ag) for processing and presentation by professional Ag-presenting cells (APC) remains limited. This represents, indeed, a bottleneck in development of vaccines inducing specific T-cell responses. Here, we describe a novel strategy of Ag delivery into APCs. The Ag of choice is fused to the N- or C-terminus of streptavidin (SA) and tetrameric Ag-SA or SA-Ag fusion proteins are produced in E. coli and purified by 2-Iminobiotin-Agarose affinity chromatography. Alternatively, Ag-SA proteins are purified from urea extracts of E. coli inclusion bodies and refolded in vitro into functional tetramers. Complexes with biotinylated antibodies targeting cell surface receptors are formed and used to deliver the Ags of choice for processing and presentation by APCs and induction of Ag-specific CD4+ and CD8+ T-cell responses in vitro and in vivo.

• MeSH
• antigeny CD11c imunologie   metabolismus   MeSH
• antigeny chemie   genetika   imunologie   metabolismus   MeSH
• biotinylace MeSH
• buněčné linie MeSH
• dendritické buňky imunologie   metabolismus   MeSH
• kur domácí MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• ovalbumin chemie   genetika   imunologie   metabolismus   MeSH
• receptory buněčného povrchu imunologie   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   imunologie   metabolismus   MeSH
• rozpustnost MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• streptavidin chemie   genetika   imunologie   metabolismus   MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH