Raman micro-spectroscopy technique offers a combination of relatively high spatial resolution with identification of components or mixtures of components in different sample areas, e.g. on the surface or the cross-section of a sample. This study is focused on the analysis of the tablets from pharmaceutical development with different technological parameters: (1) the manufacturing technology, (2) the particle size of the input API (active pharmaceutical ingredient) and (3) the quantitative composition of the individual excipients. These three mentioned parameters represent the most frequently solved problems in the field of reverse engineering in pharmacy. The investigation aims to distinguish tablets with the above-described technological parameters with limited subjective steps by Raman microscopy. Furthermore, non-subjective methods of Raman data analysis using advanced statistical analysis have been proposed, namely Principal Component Analysis, Soft Independent Modelling of Class Analogy and Linear Discriminant Analysis. The methods successfully distinguished and identified even very small differences in the analysed tablets within our study and provided objective statistic evaluation of Raman maps. The information on component and particle size distribution including their small differences, which is the critical parameter in the development of the original and generic products, was obtained due to combination of these methods. Even though each of these chemometric methods evaluates the data set from a different perspective, their mutual application on the problem of Raman maps evaluation confirmed and specified results on level that would be unattainable with the use of only one them.

• MeSH
• chemometrika MeSH
• farmacie * MeSH
• léčivé přípravky * MeSH
• pomocné látky MeSH
• Ramanova spektroskopie MeSH
• tablety MeSH
• Publikační typ
• časopisecké články MeSH

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

• MeSH
• Aquifex enzymologie   genetika   MeSH
• bakteriální proteiny chemie   genetika   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem chemie   genetika   MeSH
• katalytická doména MeSH
• katalýza MeSH
• mutageneze cílená MeSH
• sekundární struktura proteinů MeSH
• substituce aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fibrin plays an important role during wound healing and skin regeneration. It is often applied in clinical practice for treatment of skin injuries or as a component of skin substitutes. We prepared electrospun nanofibrous membranes made from poly(l-lactide) modified with a thin fibrin nanocoating. Fibrin surrounded the individual fibers in the membrane and also formed a thin fibrous mesh on several places on the membrane surface. The cell-free fibrin nanocoating remained stable in the cell culture medium for 14 days and did not change its morphology. On membranes populated with human dermal fibroblasts, the rate of fibrin degradation correlated with the degree of cell proliferation. The cell spreading, mitochondrial activity, and cell population density were significantly higher on membranes coated with fibrin than on nonmodified membranes, and this cell performance was further improved by the addition of ascorbic acid in the cell culture medium. Similarly, fibrin stimulated the expression and synthesis of collagen I in human dermal fibroblasts, and this effect was further enhanced by ascorbic acid. The expression of beta1-integrins was also improved by fibrin, and on pure polylactide membranes, it was slightly enhanced by ascorbic acid. In addition, ascorbic acid promoted deposition of collagen I in the form of a fibrous extracellular matrix. Thus, the combination of nanofibrous membranes with a fibrin nanocoating and ascorbic acid seems to be particularly advantageous for skin tissue engineering.

• MeSH
• buněčná diferenciace MeSH
• elektrochemie metody   MeSH
• extracelulární matrix metabolismus   MeSH
• fibrin chemie   metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• imunoenzymatické techniky MeSH
• kolagen genetika   metabolismus   MeSH
• kultivované buňky MeSH
• kůže cytologie   metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nanovlákna chemie   MeSH
• polyestery chemie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proliferace buněk MeSH
• regenerace fyziologie   MeSH
• tkáňové inženýrství metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nine new dihydrochloride salts of 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-alkoxyethoxybenzoates were designed and synthesized. The physicochemical properties such as lipophilicity index (log kw) and dissociation constant (pKa) were experimentally determined and compared to the software calculated data. The lipophilicity index was determined by means of reversed-phase high performance liquid chromatography (RP-HPLC). The pKa values were determined by means of capillary zone electrophoresis. The "drug-likeness" properties according to the Lipinski Rule of Five and prediction of possible blood-brain barrier penetration were computed and discussed.

• MeSH
• benzoáty chemická syntéza   chemie   MeSH
• chemické jevy MeSH
• chromatografie s reverzní fází metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• koncentrace vodíkových iontů MeSH
• lipidy chemie   MeSH
• software * MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental-in silico computational based approach was adopted for the identification and characterization of miRNAs in Humulus lupulus (hop), which is widely cultivated for use by the brewing industry and apart from, used as a medicinal herb. A total of 22 miRNAs belonging to 17 miRNA families were identified in hop following comparative computational approach and EST-based homology search according to a series of filtering criteria. Selected miRNAs were validated by end-point PCR and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), confirmed the existence of conserved miRNAs in hop. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 47 potential miRNA targets were identified in hop. Strikingly, the majority of predicted targets were belong to transcriptional factors which could regulate hop growth and development, including leaf, root and even cone development. Moreover, the identified miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and other physiological processes. The cis-regulatory elements relevant to biotic and abiotic stress, plant hormone response, flavonoid biosynthesis were identified in the promoter regions of those miRNA genes. Overall, findings from this study will accelerate the way for further researches of miRNAs, their functions in hop and shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available.

• MeSH
• databáze genetické MeSH
• exprimované sekvenční adresy * MeSH
• genové regulační sítě genetika   MeSH
• Humulus genetika   MeSH
• mikro RNA genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• regulace genové exprese u rostlin genetika   MeSH
• rostlinné geny genetika   MeSH
• software * MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial F-type adenosine triphosphate (ATP) synthases are commonly introduced as highly conserved membrane-embedded rotary machines generating the majority of cellular ATP. This simplified view neglects recently revealed striking compositional diversity of the enzyme and the fact that in specific life stages of some parasites, the physiological role of the enzyme is to maintain the mitochondrial membrane potential at the expense of ATP rather than to produce ATP. In addition, mitochondrial ATP synthases contribute indirectly to the organelle's other functions because they belong to major determinants of submitochondrial morphology. Here, we review current knowledge about the trypanosomal ATP synthase composition and architecture in the context of recent advances in the structural characterization of counterpart enzymes from several eukaryotic supergroups. We also discuss the physiological function of mitochondrial ATP synthases in three trypanosomatid parasites, Trypanosoma cruzi, Trypanosoma brucei and Leishmania, with a focus on their disease-causing life cycle stages. We highlight the reversed proton-pumping role of the ATP synthase in the T. brucei bloodstream form, the enzyme's potential link to the regulation of parasite's glycolysis and its role in generating mitochondrial membrane potential in the absence of mitochondrial DNA.

• MeSH
• genetické inženýrství * MeSH
• Leishmania enzymologie   MeSH
• membránový potenciál mitochondrií MeSH
• mitochondriální protonové ATPasy genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Trypanosoma brucei brucei enzymologie   MeSH
• Trypanosoma cruzi enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• MeSH
• chromozomy MeSH
• lidé MeSH
• nádorová transformace buněk MeSH
• nádory diagnóza   terapie   MeSH
• replikace DNA MeSH
• telomerasa fyziologie   genetika   terapeutické užití   MeSH
• telomery fyziologie   MeSH
• tkáňové inženýrství MeSH
• Check Tag
• lidé MeSH

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

• MeSH
• buněčné linie MeSH
• chemické modely MeSH
• difrakce rentgenového záření MeSH
• financování organizované MeSH
• HIV-1 enzymologie   fyziologie   genetika   MeSH
• HIV-proteasa genetika   chemie   izolace a purifikace   metabolismus   MeSH
• inhibitory reverzní transkriptasy chemie   MeSH
• inzerční mutageneze MeSH
• katalýza MeSH
• kinetika MeSH
• konsenzuální sekvence MeSH
• látky proti HIV terapeutické užití   MeSH
• ledviny cytologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• replikace viru MeSH
• RNA virová analýza   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence MeSH
• Check Tag
• lidé MeSH

Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments. To extract this information, we propose fitting both first and second scans simultaneously. Four most common single-peak transition models are considered: (i) fully reversible, (ii) fully irreversible, (iii) partially reversible transitions, and (iv) general three-state models. The method is validated using calorimetry data for chicken egg lysozyme, mutated Protein A, three wild-types of haloalkane dehalogenases, and a mutant stabilized by protein engineering. We show that modelling of reheating increases the precision of determination of unfolding mechanisms, free energies, temperatures, and heat capacity differences. Moreover, this modelling indicates whether alternative refolding pathways might occur upon cooling. The Matlab-based data fitting software tool and its user guide are provided as a supplement.

• MeSH
• kalorimetrie metody   MeSH
• kinetika MeSH
• kuřecí embryo MeSH
• muramidasa chemie   metabolismus   MeSH
• proteinové inženýrství MeSH
• rozbalení proteinů MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3 h to 6 h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of P(NPC4):GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30 min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.

• MeSH
• Arabidopsis účinky léků   enzymologie   genetika   metabolismus   MeSH
• chlorid sodný metabolismus   farmakologie   MeSH
• fosfolipasy typu C genetika   metabolismus   fyziologie   MeSH
• geneticky modifikované rostliny MeSH
• kořeny rostlin účinky léků   enzymologie   genetika   metabolismus   MeSH
• kyselina abscisová genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH