Background: Recent evidence has shown that circulating microribonucleic acid (miRNA) has been related to many diseases either as an inhibitor or a stimulant factor, among them miRNA-122 which has proven through studies its relationship with insulin resistance, an adversative lipid profile, obesity, type 2 diabetes, and metabolic syndrome in several studies; however, the mechanisms involved are unknown. This study investigates the role of miRNA-122 expression in overweight patients suffering from metabolic disorders such as diabetes and hypertension and its relationship to the development of oxidative stress in patient groups.Materials and Methods: 30 patients with type 2 diabetes mellitus (T2DM), 30 people with hypertension (HTN), 30 patients with T2DM+HTN, and 30 healthy persons who served as controls were enrolled in this study. An ARCHITECT c4000 clinical chemistry analyzer was used to assess lipid profiles. The sandwich immunodetection approach was used to assess whole blood hemoglobinA1c. By colorimetric methodology, catalase activity (CAT), superoxide dismutase activity (SOD), malondialdehyde (MDA) levels, and advanced oxidation protein products (AOPPs) levels were measured. The expression of serum miRNA-122 was determined using the quantitative polymerase chain reaction.Results: The activity of SOD and CAT in patient groups was found to be substantially lower than in the control group (p < 0.05), whereas MDA and AOPP concentrations were found to be significantly higher in patient groups compared to the control group (p < 0.05). When patient groups were compared to control groups, the miRNA-122 level was higher in the patients (p< 0.05).Conclusions: miRNA-122 expression is involved in the pathogenesis of T2DM and HTN-induced oxidative stress, there is a reciprocal relationship between the increase in gene expression of the miRNA-122 and the increase in oxidative stress accompanied by a decrease in the effectiveness of antioxidant enzymes, which leads to the development of the disease.
- MeSH
- Blood Chemical Analysis methods instrumentation statistics & numerical data MeSH
- Biomarkers blood MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Malondialdehyde blood MeSH
- Metabolic Diseases * blood pathology MeSH
- MicroRNAs * blood MeSH
- Oxidative Stress MeSH
- Advanced Oxidation Protein Products blood MeSH
- Superoxide Dismutase blood MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Publication type
- Clinical Study MeSH
Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 μM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 μM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 μM) compared to 11b-f (GI50 = 6.4-11.5 μM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.
- Publication type
- Journal Article MeSH
Control mechanisms of spindle assembly and chromosome segregation are vital for preventing aneuploidy during cell division. The mammalian germ cells and embryos are prone to chromosome segregation errors, and the resulting aneuploidy is a major cause of termination of development or severe developmental disorders. Here we focused on early mouse embryos, and using combination of methods involving microinjection, immunodetection and confocal live cell imaging, we concentrated on the Spindle Assembly Checkpoint (SAC) and Anaphase Promoting Complex/Cyclosome (APC/C). These are two important mechanisms cooperating during mitosis to ensure accurate chromosome segregation, and assessed their activity during the first two mitoses after fertilization. Our results showed, that in zygotes and 2-cell embryos, the SAC core protein Mad1 shows very low levels on kinetochores in comparison to oocytes and its interaction with chromosomes is restricted to a short time interval after nuclear membrane disassembly (NEBD). Exposure of 2-cell embryos to low levels of spindle poison does not prevent anaphase, despite the spindle damage induced by the drug. Lastly, the APC/C is activated coincidentally with NEBD before the spindle assembly completion. This early onset of APC/C activity, together with precocious relocalization of Mad1 from chromosomes, prevents proper surveillance of spindle assembly by SAC. The results contribute to the understanding of the origin of aneuploidy in early embryos.
- Publication type
- Journal Article MeSH
Dementia is a devastating age-related disorder. Its therapy would largely benefit from the identification of susceptible subjects at early, prodromal stages of the disease. To search for such prognostic markers of cognitive impairment, we studied spatial navigation in male BALBc vs. B6N mice in combination with in vivo magnetic resonance spectroscopy (1H-MRS). BALBc mice consistently showed higher escape latencies than B6N mice, both in the Water Cross Maze (WCM) and the Morris water maze (MWM). These performance deficits coincided with higher levels of myo-inositol (mIns) in the dorsal hippocampus before and after training. Subsequent biochemical analyses of hippocampal specimens by capillary immunodetection and liquid chromatography mass spectrometry-based (LC/MS) metabolomics revealed a higher abundance of glial markers (IBA-1, S100B, and GFAP) as well as distinct alterations in metabolites including a decrease in vitamins (pantothenic acid and nicotinamide), neurotransmitters (acetylcholine), their metabolites (glutamine), and acetyl-L-carnitine. Supplementation of low abundant acetyl-L-carnitine via the drinking water, however, failed to revert the behavioral deficits shown by BALBc mice. Based on our data we suggest (i) BALBc mice as an animal model and (ii) hippocampal mIns levels as a prognostic marker of mild cognitive impairment (MCI), due to (iii) local changes in microglia and astrocyte activity, which may (iv) result in decreased concentrations of promnesic molecules.
- Publication type
- Journal Article MeSH
Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo-devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo-devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-α-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.
- Publication type
- Journal Article MeSH
Proper histological evaluation of lung tissue and accurate antigen detection by immunodetection techniques require histological tissue processing to preserve tissue reactivity and open alveolar spaces. In this study, we focused on testing and comparing different procedures of tissue cryopreservation. Sucrose or Tissue Tek were used with several methods of freezing samples by supercooled liquids and liquid nitrogen. Changes in tissue caused during the freezing of samples and the effect of cryoprotectants on the tissue were recorded. Rat and porcine pulmonary tissues were used in this experiment. This study aimed to optimize the process of lung cryopreservation with emphasis on enabling proper anatomical evaluation and preserving a high tissue immunoreactivity. The best results were obtained by inflating pulmonary parenchyma with a 1 : 1 mixture of O.C.T. with phosphate buffered saline (PBS) frozen in supercooled n-heptane placed on dry ice. Pulmonary tissue prepared in this way enables to perform proper histological evaluation and to detect target molecules by immunohistochemical analysis.
Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.
- MeSH
- Antigens, Surface analysis MeSH
- Cell Membrane radiation effects ultrastructure MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Microscopy, Atomic Force MeSH
- Nanostructures * MeSH
- Surface Properties * MeSH
- Hot Temperature * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Projekt je zaměřen na detailní analýzu aktivity RTK a navazujících signálních drah u vysoce rizikových refrakterních solidních nádorů dětského věku. Do studie budou zařazeny jednotlivé i párové vzorky nádorové tkáně odebírané pacientům. Nativní nebo zamražená tkáň bude využita pro proteinové arrays a odpovídající fixovaná tkáň zalitá v parafínových blocích bude určena pro imunodetekce. Experimentální přístupy zahrnují fosforylační proteinové arrays pro získání profilu aktivace jednotlivých RTK a navazujících signálních drah v nádorové tkáni; pro ověření aktivity kandidátních kináz pak budou využity PCR a imunodetekční metody. Získané výsledky budou především znamenat zcela nové poznatky týkající se změn v aktivitě RTK a navazujících signálních drah, které mohou být spojeny s průběhem onemocnění a mohou být důležité jako možné cíle pro léčbu nízkomolekulárními inhibitory.; This project is aimed at a detailed analysis of activity of the RTK and the downstream signaling pathways in high-risk refractory pediatric solid tumors. Individual or paired samples of tumor tissue taken from the individual patients should be included in this study. The native or fresh-frozen tumor samples should be used for phospho-protein arrays and formalin-fixed paraffin-embedded tumor samples for immunodetection analyses. The experimental approaches include the phosphorylation protein arrays to determine patterns of RTK and downstream kinase activity, the PCR and immunodetection methods to verify results obtained using protein arrays and to evaluate the expression profiles of the candidate kinases. The obtained data should bring especially completely new information concerning the changes in RTK and downstream signaling activity patterns associated with the course of disease in these patients that may be important as possible targets for new small-molecule inhibitors.
- MeSH
- Child MeSH
- Phosphorylation MeSH
- Immunologic Techniques MeSH
- Humans MeSH
- Biomarkers, Tumor MeSH
- Neoplasms therapy MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Risk Factors MeSH
- Signal Transduction MeSH
- Receptor Protein-Tyrosine Kinases MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Conspectus
- Pediatrie
- NML Fields
- onkologie
- pediatrie
- biochemie
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
Rheophytism is extremely rare in the Utricularia genus (there are four strictly rheophytic species out of a total of about 260). Utricularia neottioides is an aquatic rheophytic species exclusively growing attached to bedrocks in the South American streams. Utricularia neottioides was considered to be trap-free by some authors, suggesting that it had given up carnivory due to its specific habitat. Our aim was to compare the anatomy of rheophytic U. neottioides with an aquatic Utricularia species with a typical linear monomorphic shoot from the section Utricularia, U. reflexa, which grows in standing or very slowly streaming African waters. Additionally, we compared the immunodetection of cell wall components of both species. Light microscopy, histochemistry, scanning, and transmission electron microscopy were used to address our aims. In U. neottioides, two organ systems can be distinguished: organs (stolons, inflorescence stalk) which possess sclerenchyma and are thus resistant to water currents, and organs without sclerenchyma (leaf-like shoots), which are submissive to the water streaming/movement. Due to life in the turbulent habitat, U. neottioides evolved specific characters including an anchor system with stolons, which have asymmetric structures, sclerenchyma and they form adhesive trichomes on the ventral side. This anchor stolon system performs additional multiple functions including photosynthesis, nutrient storage, vegetative reproduction. In contrast with typical aquatic Utricularia species from the section Utricularia growing in standing waters, U. neottioides stems have a well-developed sclerenchyma system lacking large gas spaces. Plants produce numerous traps, so they should still be treated as a fully carnivorous plant.
BACKGROUND AND AIMS: Carnivorous plants can enhance photosynthetic efficiency in response to prey nutrient uptake, but the underlying mechanisms of increased photosynthesis are largely unknown. Here we investigated photosynthesis in the pitcher plant Nepenthes × ventrata in response to different prey-derived and root mineral nutrition to reveal photosynthetic constrains. METHODS: Nutrient-stressed plants were irrigated with full inorganic solution or fed with four different insects: wasps, ants, beetles or flies. Full dissection of photosynthetic traits was achieved by means of gas exchange, chlorophyll fluorescence and immunodetection of photosynthesis-related proteins. Leaf biochemical and anatomical parameters together with mineral composition, nitrogen and carbon isotopic discrimination of leaves and insects were also analysed. KEY RESULTS: Mesophyll diffusion was the major photosynthetic limitation for nutrient-stressed Nepenthes × ventrata, while biochemistry was the major photosynthetic limitation after nutrient application. The better nutrient status of insect-fed and root-fertilized treatments increased chlorophyll, pigment-protein complexes and Rubisco content. As a result, both photochemical and carboxylation potential were enhanced, increasing carbon assimilation. Different nutrient application affected growth, and root-fertilized treatment led to the investment of more biomass in leaves instead of pitchers. CONCLUSIONS: The study resolved a 35-year-old hypothesis that carnivorous plants increase photosynthetic assimilation via the investment of prey-derived nitrogen in the photosynthetic apparatus. The equilibrium between biochemical and mesophyll limitations of photosynthesis is strongly affected by the nutrient treatment.
- MeSH
- Chlorophyll MeSH
- Photosynthesis * MeSH
- Plant Leaves MeSH
- Carnivory * MeSH
- Organic Chemicals MeSH
- Carbon Dioxide MeSH
- Nutrients MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
