Michal M. Godlewski

Subcellular redistribution of BAX during apoptosis induced by anticancer drugs.

BAX is the 192-amino acid, 21-kDa protein which is ubiquitously distributed in normal tissues and is regarded as a tumor suppressor sensitizing malignant cells to anticancer drugs. In spite of many studies, the molecular mechanism of BAX action is still obscure. In the present study subcellular BAX translocations in human colon adenocarcinoma COLO 205 cells exposed to various anticancer drugs [camptothecin (CPT), etoposide (ETO), staurosporine (STP), 2-chloro-2′-deoxyadenosine (2CdA) and nimesulide (NIM)] was examined. Cells were grown on coverslips under optimal conditions (10% FCS/DMEM) or were stimulated to apoptosis with the drugs examined. Laser scanning cytometry was applied for the quantitative analysis of BAX expression, and distribution in the cytoplasmic (BAX Cf) and nuclear (BAX Nf) area. BAX maximal pixel (BAX MP), the parameter corresponding to aggregation of BAX in the cell, was also measured. All examined drugs increased the number of cells with high BAX MP, reaching the peak at 60 min after drug administration. The most pronounced effect was in the case of 2CdA, CPT and STP. The increase in BAX MP was observed only when antibody recognizing the 43-61 amino acid sequence was used. When antibody binding the N-terminal epitope (11-30 amino acid sequence) was applied, the number of cells expressing high BAX MP significantly decreased. These results indicate that apoptotic stimuli delivered by anticancer drugs led to aggregation of BAX in cancer cells, which is dependent on BAX activation by its cleavage at the N-terminal epitope and exposure of the BH3 domain. It was shown that BAX Nf increased in cells treated with CPT, STP, ETO, 2CdA and NIM, whereas BAX Cf rose after STP and NIM. The increase in BAX Nf and, occurring in most treatments, the increase in the BAX Nf:Cf ratio indicates a BAX shift from the cytoplasm to the nucleus. Furthermore, staining with different antibodies showed that only the activated form of BAX was translocated to the nucleus. Immunoelectron microscopy revealed that CPT-induced apoptosis was associated with translocation of BAX from the cytosol to organellar membranes (mitochondrial, Golgi apparatus and endoplasmic reticulum) and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. The subcellular translocations of BAX preceded in time the appearance of morphological symptoms of apoptosis. In conclusion, (i) in spite of different molecular mechanisms of apoptosis induction by the anticancer drugs examined, BAX remains a common link in the chain of reactions leading to cell death, and (ii) BAX activation and subcellular translocations from the cytosol to organellar membranes and nucleus are key cellular responses to drugs bearing proapoptotic properties.

EXPRESSION OF BAX IN CELL NUCLEUS AFTER EXPERIMENTALLY INDUCED APOPTOSIS REVEALED BY IMMUNOGOLD AND EMBEDMENT‐FREE ELECTRON MICROSCOPY

The unique combination of immunocytochemistry with embedment‐free electron microscopy was applied for precise and specific localisation of BAX in the human colon adenocarcinoma COLO 205 cell line stimulated to undergo apoptosis by camptothecin (DNA topoisomerase I inhibitor). Camptothecin‐induced apoptosis was associated with redistribution of BAX from cytosol to organelle membranes: mitochondria, Golgi apparatus, endoplasmic reticulum and via nuclear envelope pores to the nucleus, occurring within 60–180min of cell exposure to the drug. An increase in BAX immunoreactivity on fine filaments and the lamina‐pore complex of the nuclear matrix was also observed. The increase in BAX expression in the nuclear area of camptothecin‐treated COLO 205 cells was confirmed by quantitative analysis using laser scanning cytometry. The subcellular translocations of BAX preceded the appearance of any morphological symptoms of apoptosis.

Intrauterine growth retarded piglet as a model for humans--studies on the perinatal development of the gut structure and function.

The overall acceptance of pig models for human biomedical studies is steadily growing. Results of rodent studies are usually confirmed in pigs before extrapolating them to humans. This applies particularly to gastrointestinal and metabolism research due to similarities between pig and human physiology. In this context, intrauterine growth retarded (IUGR) pig neonate can be regarded as a good model for the better understanding of the IUGR syndrome in humans. In pigs, the induction of IUGR syndrome may include maternal diet intervention, dexamethasone treatment or temporary reduction of blood supply. However, in pigs, like in humans, circa 8% of neonates develop IUGR syndrome spontaneously. Studies on the pig model have shown changes in gut structure, namely a reduced thickness of mucosa and muscle layers, and delayed kinetic of disappearance of vacuolated enterocytes were found in IUGR individuals in comparison with healthy ones. Functional changes include reduced dynamic of gut mucosa rebuilding, decreased activities of main brush border enzymes, and changes in the expression of proteins important for carbohydrate, amino acids, lipid, mineral and vitamin metabolism. Moreover, profiles of intestinal hormones are different in IUGR and non-IUGR piglets. It is suggested that supplementation of the mothers during the gestation and/or the IUGR offspring after birth can help in restoring the development of the gastrointestinal tract. The pig provides presumably the optimal animal model for humans to study gastrointestinal tract structure and function development in IUGR syndrome.

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells

Smac/DIABLO, a pro‐apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT‐induced apoptosis in living MCF‐7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m‐calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax‐dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.

Co-localization of apoptosis-regulating proteins in mouse mammary epithelial HC11 cells exposed to TGF-β1

TGF-beta1 is an apoptogenic agent for mammary epithelial cells (MEC). The molecular mechanism of the TGF-beta1-induced apoptosis remains, however, obscure. In the present study we used laser scanning cytometry, confocal microscopy and immunogold electron microscopy to analyze the expression, aggregation and co-localization of caspase-8, Bid, Bax and VDAC-1. These proteins are regarded as the most important factors involved in the regulatory phase of TGF-beta1-induced apoptosis. Apoptosis in HC11 mouse MEC manifested with a simultaneous increase in expression and subcellular aggregation of caspase-8, Bid, Bax and VDAC-1. Confocal microscopy revealed a strong pattern of co-localization of examined proteins during both early and late apoptosis. Experiments with double- and triple-staining immunoelectron microscopy showed a co-localization of Bax/Bid, caspase-8/Bax/Bid, and Bax/VDAC-1, on the membranes of mitochondria, Golgi apparatus, rough endoplasmic reticulum, nuclear envelope, nuclear pore, and within the nucleus. In conclusion, the observed pattern of changes in aggregation and subcellular localization of caspase-8, Bid, Bax and VDAC-1 during TGF-beta1-induced apoptosis in HC11 mouse MEC suggests an interaction between these proteins and formation of multimeric complexes on organellar membranes, thus controlling their permeability for intracellular mediators of apoptosis.

Biodegradation of the ZnO:Eu nanoparticles in the tissues of adult mouse after alimentary application

Biodegradable zinc oxide nanoparticles (ZnO NPs) are considered promising materials for future biomedical applications. To fulfil this potential, biodistribution and elimination patterns of ZnO NPs in the living organism need to be resolved. In order to investigate gastrointestinal absorption of ZnO NPs and their intra-organism distribution, water suspension of ZnO or fluorescent ZnO:Eu (Europium-doped zinc oxide) NPs (10mg/ml; 0.3ml/mouse) was alimentary-administered (IG: intra-gastric) to adult mice. Internal organs collected at key time-points after IG were evaluated by AAS for Zn concentration and analysed by cytometric techniques. We found that Zn-based NPs were readily absorbed and distributed (3 h post IG) in the nanoparticle form throughout the organism. Results suggest, that liver and kidneys were key organs responsible for NPs elimination, while accumulation was observed in the spleen and adipose tissues. We also showed that ZnO/ZnO:Eu NPs were able to cross majority of biological barriers in the organism (including blood-brain-barrier).

Impact of yttria stabilization on Tb3+ intra-shell luminescence efficiency in zirconium dioxide nanopowders

This paper reports the observation of Tb(3+) 4f-4f emission gain in ZrO2 nanocrystals stabilized by Y2O3 as the amount of stabilizer increases from 0% to 10% mol. The nanocrystals were obtained via microwave solvothermal technology. The photoluminescence properties of as-grown samples are investigated. The possibility of biological applications of the material is tested on living organisms (mice). The result indicates the potential use of the studied material as a luminescent nanomarker.

High-k oxides by atomic layer deposition—Applications in biology and medicine

Wide band gap oxides grown by atomic layer deposition (ALD) are intensively studied for applications as insulators (gate oxides in field effect transistors) and as resistance switching materials in electronic memories. For these applications, compositions of dielectric films (laminar structures of HfO2, TiO2, ZrO2, and Al2O3) can also be used. Our research shows that the same layers show antibacterial activity comparable to ZnO. For mentioned application, it is important that the ALD allows uniform coating of various materials, including temperature sensitive ones (e.g., perishable fabrics) and equipment and instruments (including implants) used in hospitals and in a food industry. Here, the authors present the antibacterial activity of ALD-deposited metal oxides, promising a range of potential applications in medicine, veterinary, broader health care, and food industry.

Effect of butyrate infusion into the rumen on butyrate flow to the duodenum, selected gene expression in the duodenum epithelium, and nutrient digestion in sheep

The aim of the study was to determine the effect of butyrate infusion into the rumen on butyrate flow to the duodenum, expression of short-chain fatty acid (SCFA) transporters (monocarboxylate transporter-1, -2, and -4) and receptors (G protein coupled receptor-41 and -43) in the duodenal epithelium and nutrient digestion in sheep. Eight wethers (39.0 ± 3.00 kg; mean ± SD) with ruminal and T-shape duodenal cannulas were allocated to 4 × 4 replicated Latin square design with each experimental period lasting for 21 d (12 d of adaptation and 9 d for data and sample collection). Experimental treatments were: 1) distilled water infusion into the rumen (CONT); 2) 15 g/d of butyric acid infusion into the rumen (BUT15); 3) 30 g/d of butyric acid infusion into the rumen (BUT30); and 4) 45 g/d of butyric acid infusion into the rumen (BUT45). The daily dose of butyrate was infused into the rumen via the rumen cannula, with 200 mL of solution of butyric acid and distilled water, at a constant rate (0.1389 mL/min) throughout the day using a peristaltic pump. Correspondingly, 200 mL/d of distilled water was infused into the rumen of CONT. The wethers were fed daily 900 g of chopped meadow hay and 200 g of concentrate in two equal meals at 0600 and 1800 h. Butyrate infusion into the rumen did not affect total SCFA concentration in the rumen fluid ( > 0.11). Molar proportion of butyrate in total SCFA linearly increased, and molar proportion of acetate and isovalerate linearly decreased ( ≤ 0.02) with an increasing amount of butyrate infused into the rumen. The molar proportion of butyrate in total SCFA in the duodenal digesta linearly increased ( < 0.01), and butyrate flow to duodenum tended to linearly increase ( = 0.06) with an increasing dose of exogenous butyrate delivered to the rumen. Butyrate infusion into the rumen did not affect ( ≥ 0.14) the mRNA expression of monocarboxylate transporter-2 and -4 and G protein coupled receptor-43 in the duodenal epithelium. The G protein coupled receptor-41 and monocarboxylate transporter-1 mRNA expression in the duodenal epithelium was very low in many of the analyzed samples. Digestibility of organic matter, neutral detergent fiber, and acid detergent fiber in the stomach (forestomach and abomasum) decreased for BUT15 and BUT30 and then increased for BUT45 (quadratic, ≤ 0.04); however, neither digestibility in the intestine nor total tract digestibility differed between treatments ( ≥ 0.10).

Minute kinetics of proapoptotic proteins: BAX and Smac/DIABLO in living tumor cells revealed by homeostatic confocal microscopy

Traditional methods of visualization and analysis based on fixed cell populations treated with the drug for a different time give the limited possibility of time-sequence analysis. In time-lapse microscopy where the whole cell is observed regardless to intracellular structure, precise localization of events and differentiation between colocalization and overlapping of the fluorescence is impossible. Furthermore prolonged experiments with living cells increased the influence of improper environmental conditions. Homeostatic confocal microscopy gives an exceptional insight into minute pattern of changes occurring in the same living cell maintained in stable conditions during whole experimental period. It is built on a confocal system equipped with the homeostatic chamber providing constant, monitored heating and moisturized, CO2-enriched atmosphere during long period observations. In the present study 2D/time and 4D homeostatic confocal microscopy were applied for analysis of minute pattern of changes occurring at the mitochondria. The release of Smac/DIABLO from mitochondria in tumor cells under the apoptogenic stimulus, consist of two phases: the first immediately after drug administration, and the major second one after 15 min. Furthermore the time-pattern of BAX translocation to the mitochondria and Smac/DIABLO release coincide, suggesting that the release of Smac/DIABLO is correlated with BAX translocation to the mitochondria.