Mechanisms involved in the development of the small intestine mucosal layer in postnatal piglets.

The use of complementary visualization and measurement techniques allowed accurate description and quantification of changes in the intestinal mucosal architecture and provided a comprehensive outlook on the dynamics of remodelling and maturation processes of the mucosal layer taking place in the small intestine of piglets from birth to weaning. The aim of the study was to examine the early postnatal development of the small intestine in pigs. Three techniques were used: scanning electron microscopy (measurements of villus density and shape, height of enterocytes and microvilli, cell exfoliation, and location of extrusion zones), optical microscopy (cross section, measurement of structures: villus length and width; crypt depth; mucosal thickness), and confocal microscopy (cell localization, apoptosis, exfoliation and migration). The postnatal development of the mucosal layer of the small intestine was reflected in changes in the density, length, width, and shape of villi, crypt depth, replacement of enterocyte population, and arrangement. The presence of deep transverse furrows on villus corpus and vacuolated fetal-type enterocytes in the mucosal layer of the small intestine, which are able to engulf large amounts of colostrum shortly after birth, appears to play an important role in the observed phenomenon of straightening of the villus height and increasing of the villus diameter shortly after birth. We hypothesized that the intestinal mucosal layer is compressed before birth and ready to unfold within a short time after birth.

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).

Application of scanning cytometry and confocal-microscopy-based image analysis for investigation the role of cytoskeletal elements during equine herpesvirus type 1 (EHV-1) infection of primary murine neurons.

Equine herpesvirus type 1 (EHV-1), a member of Alphaherpesvirinae, has a broad host range in vitro, allowing for study of the mechanisms of productive viral infection, including intracellular transport in various cell cultures. In the current study, quantitative methods (scanning cytometry and real-time PCR) and confocal-microscopy-based image analysis were used to investigate the contribution of microtubules and neurofilaments in the transport of virus in primary murine neurons separately infected with two EHV-1 strains. Confocal-microscopy analysis revealed that viral antigen co-localized with the ß-tubulin fibres within the neurites of infected cells. Alterations in ß-tubulin and neurofilaments were evaluated by confocal microscopy and scanning cytometry. Real-time PCR analysis demonstrated that inhibitor-induced (nocodazole, EHNA) disruption of microtubules and dynein significantly reduced EHV-1 replication in neurons. Our results suggest that microtubules together with the motor protein - dynein, are involved in EHV-1 replication process in neurons. Moreover, the data presented here and our earlier results support the hypothesis that microtubules and actin filaments play an important role in the EHV-1 transport in primary murine neurons, and that both cytoskeletal structures complement each-other.

Bone marrow-origin stem/progenitor cells in the mammary gland of heifers.

The aim of the study was to estimate the size of bone marrow-origin stem/progenitor population in 2-year old nonpregnant Holstein-Friesian heifers. Quantitative and qualitative analysis was done using scanning cytometry and confocal microscopy of mammary tissue slices labelled with the combination of two markers: Sca-1 (marker of stem-progenitor cells) and CD45 (marker of hematopoietic cells). The average (+/- SEM) percentage of Sca-1POS CD45 POS cells was 0.89 +/- 0.21. They were localized mainly outside of mammary ducts, in the stroma and sometimes intraluminally. Our results indicate that the subpopulation of Sca-1POS cells bearing CD45 antigen may enrich the niche of mammary stem/progenitor cells from the bone marrow and participate in the growth of the mammary gland in post-pubertal heifers.

Equine herpesvirus type 1 (EHV-1)-induced rearrangements of actin filaments in productively infected primary murine neurons.

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. In the present study, we investigated reorganization of the cytoskeleton in neurons infected with two EHV-1 strains: Jan-E (wild-type strain) and Rac-H (attenuated strain). The studies were performed on primary murine neurons, which are an excellent model for studying neurotropism and neurovirulence of EHV-1. We have demonstrated for the first time that EHV-1 infection causes rearrangements in the actin network of neurons that are dependent on the virus strain and its adaptation to cell culture in vitro. Immunofluorescent labeling and confocal microscopy revealed the formation of long, thin projections in neurons infected with the Jan-E strain, which was probably associated with enhanced intracellular spread of the virus. The EHV-1 Rac-H strain caused disruption of the microfilaments system and general depolymerization of actin, but treatment of neurons with cytochalasin D or latrunculin A resulted in limitation of viral replication. It can therefore be assumed that actin filaments are required only at the early stages of infection. Our results allow us to suggest that the actin cytoskeleton participates in EHV-1 infection of primary murine neurons but is not essential, and that other components of the cytoskeleton and/or cellular mechanisms may be also involved during EHV-1 infection.

Influence of mutation in cj0183 and cj0588 genes for colonization abilities of Campylobacter jejuni in Caco-2 cells using confocal laser scanning microscope.

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with homology to virulence factors found in other bacteria. Previous studies showed that single mutation in the cj0183 gene does not affect adhesion of C. jejuni to the Caco-2 cell line whereas protein encoded by cj0588 is involved in adherence to the Caco-2 cells. In the presented study differences in invasion index were observed between mutants in both genes and single mutation of cj0588 in 81116 and 81-176 C. jejuni strains This fact indicates that Cj0183 protein might play some role in invasion of bacteria into host cells.

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.

Apoptotic and necrotic changes in cultured murine neurons infected with equid herpesvirus 1.

Equid herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae, is a neurotropic virus, that causes latent infections in the nervous system of the natural host. All alphaherpesviruses have developed sophisticated strategies to interfere with the host cell apoptotic mechanisms, but the ability of EHV-1 to induce apoptosis in neurons has not been determined yet. In this study, apoptotic and necrotic changes in cultured murine neurons were methods identifying key stages of apoptosis. These methods have demonstrated characteristic apoptosis features, like DNA fragmentation, chromatin condensation, membrane blebbing and cell shrinkage in the infected cells. It seems likely that apoptosis was the predominant way of cell death in EHV-1-infected murine neurons. However, we showed also that during acute EHV-1 infection the majority of infected neurons remained unchanged and survived for more than eight weeks in culture, suggesting some protective mechanisms induced by the virus. Furthermore, it was shown that infection of neurons with EHV-1 has no significant influence on the level of the caspase 3, 7, and 8. We speculate that the control of apoptosis may be the key mechanism regulating the balance between productive and latent infection at the site of virus persistence.

Opposite effects of two different strains of equine herpesvirus 1 infection on cytoskeleton composition in equine dermal ED and African green monkey kidney Vero cell lines: application of scanning cytometry and confocal-microscopy-based image analysis in a quantitative study.

Viruses can reorganize the cytoskeleton and restructure the host cell transport machinery. During infection viruses use different cellular cues and signals to enlist the cytoskeleton for their mission. However, each virus specifically affects the cytoskeleton structure. Thus, the aim of our study was to investigate the cytoskeletal changes in homologous equine dermal (ED) and heterologous Vero cell lines infected with either equine herpesvirus 1 (EHV-1) strain Rac-H or Jan-E. We found that Rac-H strain disrupted actin fibers and reduced F-actin level in ED cells, whereas the virus did not influence Vero cell cytoskeleton. Conversely, the Jan-E strain induced polymerization of both F-actin and MT in Vero cells, but not in ED cells. Confocal-microscopy analysis revealed that alpha-tubulin colocalized with viral antigen in ED cells infected with either Rac-H or Jan-E viruses. Alterations in F-actin and alpha-tubulin were evaluated by confocal microscopy, Microimage analysis and scanning cytometry. This unique combination allowed precise interpretation of confocal-based images showing the cellular events induced by EHV-1. We conclude that examination of viral-induced pathogenic effects in species specific cell lines is more symptomatic than in heterologous cell lines.

Combination of hydroxyapatite islets with Ti3P surface layer produced on titanium alloy for bone implants.

This study is concerned with the properties and bioactivity and biocompatibility of hydroxyapatite islets deposited on a new composite layer Ti3P+Ti2Ni type produced by a duplex method on Ti6Al4V titanium alloy. The microstructure and chemical composition of a produced surface layers and hydroxyapatite coating were investigated using scanning electron microscope equipped with EDS. Their bioactivity were examined in simulated body fluid and analyzed with XPS. Dissolution of hydroxyapatite was tested in culture medium during 12 days of incubation. Biocompatibility was investigated in osteoblast Saos2 line culture in contact with the tested material. Cell proliferation and activity were determined by the MTT test and measurement of alkaline phosphatase activity, respectively. Cell distribution was analyzed under a confocal microscope. The produced surface layers have a diffusion character with fine-grained structure and about 4 microm thick external zone of Ti3P. The experiments revealed higher bioactivity and biocompatibility of the Ti3P in comparison with reference titanium alloy. Hydroxyapatite islets were 0.8 mm in diameter and about 300 nm thick. They partially dissolved during the experiment what lead to formation on Ti3P between hydroxyapatite islets a precipitate containing Ca and P. Biocompatibility analyzed under confocal microscope in range of cell adhesion with osteoblast cells of Saos2 line revealed initial the highest osteoblast adhesion on Ti3P between hydroxyapatite islets and increasing on hydroxyapatite during following days. Cell were characterized by high proliferation and ALP activity. Therefore, the high bioactivity and biocompatibility of Ti3P and profitable hydroxyapatite properties make this composite layer promising for increasing implant fixation in vivo.

Large-conductance K+ channel openers induce death of human glioma cells.

Large-conductance Ca(2+)-activated K(+) channels (BKCa channels) are highly expressed in human glioma cells. It has been reported that BK(Ca) channels are present in the inner mitochondrial membrane of the human glioma cell line LN229. In the present study we investigated whether BK(Ca)-channel openers, such as CGS7181 (ethyl 2-hydroxy-1-[[(4-methylphenyl)amino]oxo]-6-trifluoromethyl-1H-indole-3-carboxylate) and CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate), affect the functioning of LN229 glioma cell mitochondria in situ. In the micromolar concentration range CGS7181 and CGS7184 induced glioma cell death. Morphological and cytometric analyses confirmed that both substances trigger the glioma cell death. This effect was not inhibited by the pan-caspase inhibitor z-VAD-fmk. Lack of DNA laddering, PARP cleavage, and caspase 3 activation suggested that glioma cell death was not of the apoptotic type. We examined the effect of CGS7184 on mitochondrial membrane potential and mitochondrial respiration. Potassium channel opener CGS7184 increased cell respiration and induced mitochondrial membrane depolarization. The latter was dependent on the presence of Ca(2+) in the external medium. It was shown that CGS7184 induced an increase of cytosolic Ca(2+) concentration due to endoplasmic reticulum store depletion. In conclusion, our results show that CGS7181 and CGS7184 induce glioma cell death by increasing the cytosolic calcium concentration followed by activation of calpains.

Control of development of gastrointestinal system in neonates.

Our recent studies of structure and function of gastrointestinal tract mucosa revealed that the domestification of Sus scrofa corresponds with the significant slowing of the organ development. On top of genetic potential, the nutritional factors (or more precisely - lack of certain biologically active substances in the feed of pregnant sows) are responsible. Moreover, feeding neonates with milk replacers instead of mother's milk further slows down the development. This is manifested by reduced mitotic activity in the crypts and enhanced apoptosis of enterocytes. The negative effects consist of slower replacement of fetal type, vacuolated enterocytes to adult type enterocytes, modified profile of brush border enzymes, alterations in intestinal mucosa barrier, higher susceptibility to infectious agents, and many others. On the other hand, farmers in order to intensify the production, shorten the suckling period imposing the neonatal piglets to be weaned at 3-4 weeks of life and even earlier. Altogether, it makes the weaning disorders one of the most important problems in pig husbandry, and the mortality of piglets in the leading pig-producing countries still reaches 10%. A number of strategies have been developed to counteract the post-weaning problems. One of them is to stimulate the development of the gastrointestinal tract of the neonate by supplementation of the sow diet with certain biologically active substances and plants. The other idea is to speed up the postnatal development of the gut mucosa for example by plant lectins. Lessons from pig studies can be also useful in human nutrition and medicine since the development of porcine gastrointestinal tract shows a great similarity to that of humans.

Molecular mechanism of programmed cell death in the gut epithelium of neonatal piglets.

In the intestinal mucosa of pig, calf and rat neonates, we observed the cells die in the packets which suggests involvement of some paracrine factors. The death signal was transferred via tissue continuum as well as across the gut lumen, and the involvement of TGF-beta1 and TNFalpha was demonstrated. Present study aimed to clarify the molecular mechanisms of programmed cell death in the mucosa of the small intestine of pig neonates. Groups (packets) of cells and the neighboring cells underwent apoptosis, and expressed an enhanced TGF-RII. In the dying cells the death signal promoted via TGF-RII was associated with enhanced expression of active caspase 8, TGF-beta1, TNFalpha and Bid. Quantitative study showed that high expression of TGF-beta1 was positively correlated with expression of BID and negatively with BCL-2, illustrating the transmission of signal from TGF-RII through SMAD cascade and RunX protein. We hypothesize that TGF-beta1 sensitizes the enterocytes for TNFalpha signaling and both cytokines control the apoptosis process in the gut epithelium. Intensive mitosis triggers many errors in DNA replication, and the role of p53 is to detect them and promote either repair or apoptosis. During first days of live all damaged cells were directed towards apoptosis while at day 7 at least some of them were repaired. Autophagy, the second form of programmed cell death, was recognized by its key marker MAP I LC3. Our data showed the colocalization of MAP I LC3 with active caspase 3 thus suggesting a coexistence between these two forms of cell death, at least in the early postnatal life.

The effect of supplementing sow with bioactive substances on neonatal small intestinal epithelium.

Development of the small intestinal epithelium in early postnatal period has a significant influence on pig's survival rate and further productivity. The aim of this research was to verify whether the diet supplementation of pregnant and lactating sow with a blend of bioactive substances (flax seed, rapeseed, linden inflorescence, taurine, L-carnitine and tocopherol acetate) had an effect on the development of intestinal epithelium in their offspring. The doses of bioactive substances were calculated to meet the demands for optimal supply of the pig fetuses and newborns. Pig neonates from two groups of sows, control and supplemented, were sacrificed at the day 1, 2, 4, 7 and 14 of life. The samples taken from mid-jejunum were evaluated for mitosis (Ki67), apoptosis (active caspase 3), autophagy (MAP I LC3), and DNA damage (p53). Increase of mitotic index was noticed at day 1, 4 and 7 for supplemented group when compared to the control. Reduction of apoptotic index was observed at day 2 as compared to control. A tendency toward elevated autophagy was observed during the first 2-4 postnatal days in both groups. p53 expression was significantly lower in supplemented group as compared to control. Overall, the mitosis to programmed cell death ratio was increased and the maturation of epithelial cells quickened. We suppose that the supplementation of pregnant and lactating sow diet with bioactive substances enhanced maturation of the small intestinal epithelium in their offspring during the early postnatal period.

Quantitative study of soybean-induced changes in proliferation and programmed cell death in the intestinal mucosa of young rats.

The use of soybean in human and animal nutrition is limited because of high content of bioactive compounds: enzyme inhibitors, polyphenols, goitrogens, phytates, saponins, sugars, and agglutinins. The damage of intestinal mucosa structure was previously observed in animals fed soybean supplemented diets. Hence, the objectives of the presented study were to compare intensity of epithelium remodeling processes in different intestinal segments, and to evaluate the influence of the 1% of soybean dietary supplementation on the processes in intestinal mucosa. The experiment was performed on 30 Wistar rats fed AIN-93 based diets. Animals were divided randomly into three groups: control (CTRL), with 1% of raw soybean (RS) and with 1% of soaked and boiled soybean (BS). The samples of: duodenum (DUO), proximal jejunum (PROX), mid-jejunum (MID), distal-jejunum (DIST) and ileum (ILE) were collected. The following processes in these samples were evaluated: mitosis (Ki-67), apoptosis (Cpp32), autophagy (MAP I LC3) and DNA damage (p53). Present data show that modification of soybean by soaking and subsequent boiling markedly influences the enterocyte turnover in the small intestine mucosa. Increased mitotic ratio in the intestine of rats fed with boiled soybean masks the negative effects of soybean on the small intestine structure.

Into the unknown--the death pathways in the neonatal gut epithelium.

Apoptosis is a fundamental process in the development of the fast growing intestinal mucosa. Apoptotic cells are present along the whole length of the villi and in the crypts. The mechanisms involved in the induction of apoptosis in the gut mucosa are still unknown. Cytokines are believed to play a role in auto- and paracrine models because the cells are dying in so-called "packets" containing neighboring cells. In the rapidly developing gut of neonates, the apoptosis rate is transiently reduced in the first days of life, enhancing the growth of mucosa. Afterwards, apoptosis plays a role in the exchange of the enterocyte population, facilitating maturation of the mucosa. The presence of autophagic cells has been confirmed for the first time in the developing gut. Deprivation of growth factors during feeding artificial milk formula led to an increased apoptosis rate. Supplementation with leptin reduced cell apoptosis and increased the mitosis-to-apoptosis ratio. Autophagy was also diminished. The key to healthy gut mucosa growth in early life, especially in fast-growing animals, is colostrum, which supplies nutritional and defensive components together with supplementary growth factors, cytokines and hormones essential for growth and maturation of gut mucosa.

Antiproliferative and apoptotic effect of TGF-beta 1 in bovine mammary epithelial BME-UV1 cells.

Transforming growth factor beta1 (TGF-beta(1)) is regarded as an important auto/paracrine regulator of mammary gland involution, however, its apoptotic effect and inhibition of growth in bovine mammary epithelial cells (MEC) has not been documented. In the present study, laser scanning cytometry, confocal and immunoelectron microscopy techniques were used for quantitative and qualitative analyzes of apoptosis, cell cycle and expression, subcellular redistribution and interactions of apoptosis-related proteins in bovine BME-UV1 MEC exposed to TGF-beta(1). TGF-beta(1) exerted both antiproliferative and apoptotic action. The antiproliferative effect was manifested by increase of cell number in G1 phase with simultaneous decrease of cell number in S and G2/M phases. It resulted in significant increase of G1/S ratio in TGF-beta(1) treated cells, indicating partial cell cycle arrest at the G1-S transition. Apoptosis induced by TGF-beta(1) manifested by characteristic morphological changes. Among biochemical features of TGF-beta(1)-induced apoptosis in BME-UV1 cells we found: (1) an increase of cell number with lowered DNA content and condensed chromatin, (2) enhanced expression of caspase-3 and m-calpain, (3) elevated number of 89 kDa PARP degradation fragments, and (4) aggregation of Bax and its interactions with voltage dependant anion channel-1. In conclusion, antiproliferative and apoptotic action of TGF-beta(1), observed in the culture of BME-UV1 cells, suggests an essential role of this cytokine in the regulation of mammary gland involution in cow.

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 60-180 min 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.

Molecular mechanism of TGF-beta1-induced apoptosis in HC11 mouse mammary epithelial cells (MEC).

The involvement of p53, Bax, cytochrome C and CPP-32 (caspase-3) in the molecular mechanism ofTGF-beta1-induced apoptosis in HC11 mouse mammary epithelial cells (MEC) was examined. Laser scanning cytometry (LSC) was applied for the quantitative analysis of expression and distribution of examined apoptosis-related proteins in the cytoplasmic (Cf) and nuclear (Nf) area. Maximal pixel of fluorescence (MP) parameter corresponding to aggregation of molecules in the cell was also measured. Confocal and immunoelectron microscopy were used as a complementary methods. Apoptosis induced by TGF-beta1 (2 ng/ml) was associated with the increase of Bax MP observed within 60 min. after cytokine administration, indicating aggregation of Bax in the cell. Immunoelectron microscopy revealed Bax aggregation on mitochondrial membranes, rough endoplasmic reticulum, Golgi apparatus, cytoskeleton, nuclear envelope and inside of nucleus. The accumulation of Bax in the nucleus was confirmed by compartmental Bax analysis, showing the increase of cell number with elevated Bax Nf in 2 hr after TGF-beta1 administration to the culture. The redistribution of Bax within the cell was dependent on its activation occurring by the cleavage at N-terminal epitope and exposure of BH3 domain. Bax aggregation on organelles was completely abolished by prolactin or IGF-I. TGF-beta1 increased p53 MP, evidently after 4 hr of cell culture exposure to this cytokine. p53 was accumulated first of all in the nucleus, which was shown by significant increase of p53 Nf/Cf ratio and increase of p53-related nuclear fluorescence on confocal images. TGF-beta1 decreased cytochrome C MP, which corresponded to its release from mitochondria and dissipation in the cytosol. It was accompanied by the increase of CPP-32 MP and concentration of 89 kDa product of PARP degradation in the nucleus. In conclusion, TGF-beta1 triggers apoptosis in MEC through mitochondrial pathway involving: activation and translocation of Bax to mitochondrial membranes, release of cytochrome C from mitochondria, activation of CPP-32 and degradation of its substrate - PARP in the nucleus. Activation and subcellular redistribution of Bax is inhibited by lactogenic hormones: prolactin and IGF-I.