Chromatin structure and the state of human organism

The state of chromatin in human buccal epithelium cell nuclei upon the influence of sport trainings was investigated. Chromatin state was evaluated in interphase buccal cell nuclei after orcein staining. The heterochromatin granule quantity (HGQ) was estimated in 30 nuclei per sample, and for every donor the mean HGQ value per 30 cells was determined. Donors of masculine sex, aged from 18 to 48 years performed training walks and samples of buccal epithelium were collected. Sportive charges induced the process of chromatin condensation in cell nuclei. After the period of repose (24-48 h) the HGQ decreased to control level therefore the process of chromatin decondensation was observed. The state of chromatin changes in connection with circadian rhythm. Chromatin became more condensed at nighttime and less condensed in the morning. Hormones such as adrenaline, noradrenaline, and hydrocortisone in vitro induced the increase of HGQ.     

Modification of electrokinetic properties of nuclei in human buccal epithelial cells by electric fields

The influence of an alternating (50 Hz) electric field (5--110 V/cm) on the state of human buccal epithelium cells was studied by the methods of intracellular microelectrophoresis, heterochromatin staining with orcein, and indigo carmine staining for viability and membrane integrity evaluations. Electric field exposure induced an increase in electrophoretic mobility of cell nuclei, decreased numbers of heterochromatin granules near the inner membrane of cell nucleus, and induced cell membrane damage; but cell viability was conserved. Nuclear and cell membrane properties varied with electric field strength and age of the donors. The data obtained are interpreted as evidence of electric field induced activation of the functional state of nuclei.     

Synergistic Effect of Betel Quid,Tobacco, and Alcohol in Cigarette Smoking Induced Micronuclei in a Population of Arunachal Pradesh,India

Genotoxicity is one of the important endpoints for risk assessment of various lifestyle factors. The present study examined the synergistic effect of tobacco, betel quid, and alcohol in cigarette smoking induced micronuclei (MN) in the buccal epithelia of exposed individuals. Analysis of MN frequency and nuclear abnormalities (binucleated, karyorrhectic, karyolitic, and pyknotic cells) was performed in the exfoliated buccal cells of 110 habituates and compared to a control group matched for gender, age, and habit. A significant increase in the frequency of MN was found in smokers and alcohol, betel quid, and tobacco users compared to the control group. Tobacco, alcohol, and betel quid seem to potentiate the effect of cigarette smoking induced MN formation in the buccal epithelium. Smoking alone significantly increased the number of karyorrhexis cells in the buccal epithelium and combined exposure of all four test substances significantly increased the number of karyorrhexis and pycnotic cells. The findings indicate a synergistic effect between smoking, betel quid, tobacco, and alcohol in MN induction and cell death in buccal cells of exposed individuals.     

Effect of the Y chromosome on the morphology of human F-chromatin under normal and pathological conditions]

Morphological peculiarities of brightly fluorescent chromatin (referred to as F-chromatin) in cell nuclei of buccal epithelium stained with propil-quinacrine mustard are studied in 94 healthy men and in 67 healthy women; in 4 men with 46,XYq--, I man with 46,XYq+; in 15 patients with the Kleinfelter syndrome (47,XXY) in 7 women with 46,XY and 5 males with 47,XYY. Diametre of F-chromatin bodies in buccal cells of healthy men varied within 0.9--0.2 mkm. Classification of types of interphase nuclei is proposed based on the rise, quantity and arrangement of F-chromatin bodies. The study of F-chromatin and X-chromatin showed independent behavior of these structures in cells of buccal smears obtained from 15 patients with the Kleinfelter syndrome.     

Changes in the electrokinetic properties of human cell nuclei during reflexotherapy

The effect of acupuncture and microwave resonance therapy (MRT) on the electrophoretic motility of the cell nuclei of buccal epithelium was studied using the electronegative nuclei index in percent (ENN index, %) during treatment of patients with a duodenal ulcer and spinal osteochondrosis. This method was developed in Kharkov State University under the direction of Prof. V.G. Shakhbazov. A special device and a chamber were used to provide the intracellular microelectrophoresis of native cell nuclei. The tested methods of reflexotherapy had the normalizing effect on the ENN index, %. Acupuncture and MRT similarly affect the human organism in terms of the tested index. These findings confirmed a direct relationship between the human health status and ENN index, % which was determined by us earlier. This method makes it possible to provide the additional monitoring of the patient’s health status.     

Spectral luminescence studies of cells stained with acrichine derivatives

Excitation and fluorescence spectra are given of quinacrine derivative solutions, of buccal epithelium cell nuclei, of peripheral blood cells, and of isolated chromosomes treated with propyl-quinacrine mustard. It is confirmed that the differential cell treatment with quinacrine derivates may be observed in aqueous solutions only. Data obtained allow us to give some recommendations for employment of optimal filters and dichroic beam-splitters in the fluorescence microscopy of chromosomes treated with quinacrine derivatives.     

Study of Y-chromatin in the buccal epithelium of man]

Investigation was carried on by fluorescent-microscopic technique, smears of buccal epithelium being stained with acrichinium-mustard gas. The extent, form, location in a nucleus and the frequency of Y-chromatin in human buccal smears were studied. In medico-genetic practice this method is employed for initial determination of chromosome sex in disease associated with anomalies of sex chromosomes.     

Changes in the expression of blood-group carbohydrates during oral mucosal development in human fetuses

Abstract. The distribution of blood group carbohydrate chains with antigen A, B, H type 2 chain (A and B precursor), and N-acetyllactosamine (H type 2 precursor) specificity was studied in human oral epithelium from different anatomical regions. These represented various epithelial differentiation patterns such as non-keratinized, parakeratinized, and orthokeratinized stratified squamous epithelium. The material included buccal and palatal epithelium from 20 persons with blood group A or O, gingival, and alveolar epithelium from 10 persons with blood group A or B, and buccal metaplastically keratinized epithelium from nine blood group A, two blood group B, and nine blood group O individuals. The blood group carbohydrate chains were examined in tissue sections by immunofluorescence microscopy. The A and B blood group antigens were detected by human blood group sera, and antigen H type 2 chains and N-acetyllactosamine by murine monoclonal antibodies. Each antigen showed a similar staining pattern in buccal and alveolar epithelium (non-keratinized) which differed considerably from that seen in palatal and gingival epithelium (ortho- and parakeratinized). The expression of blood group antigens A or B and the precursor antigen H type 2 chains in metaplastically keratinized buccal epithelium was found to differ significantly from that seen in normal non-keratinized buccal epithelium. The regional variations demonstrated in cell surface carbohydrates are suggested to reflect differences in tissue differentiation.     

Regional variations of cell surface carbohydrates in human oral stratified epithelium

The distribution of blood group carbohydrate chains with antigen A, B, H type 2 chain (A and B precursor), and N-acetyllactosamine (H type 2 precursor) specificity was studied in human oral epithelium from different anatomical regions. These represented various epithelial differentiation patterns such as non-keratinized, parakeratinized, and orthokeratinized stratified squamous epithelium. The material included buccal and palatal epithelium from 20 persons with blood group A or O, gingival, and alveolar epithelium from 10 persons with blood group A or B, and buccal metaplastically keratinized epithelium from nine blood group A, two blood group B, and nine blood group O individuals. The blood group carbohydrate chains were examined in tissue sections by immunofluorescence microscopy. The A and B blood group antigens were detected by human blood group sera, and antigen H type 2 chains and N-acetyllactosamine by murine monoclonal antibodies. Each antigen showed a similar staining pattern in buccal and alveolar epithelium (non-keratinized) which differed considerably from that seen in palatal and gingival epithelium (ortho- and parakeratinized). The expression of blood group antigens A or B and the precursor antigen H type 2 chains in metaplastically keratinized buccal epithelium was found to differ significantly from that seen in normal non-keratinized buccal epithelium. The regional variations demonstrated in cell surface carbohydrates are suggested to reflect differences in tissue differentiation.     

Effects of single-dose irradiation on bronchial epithelium: a comparison of BEAS 2B cell monolayers, human organ cultures, and Goettinger minipigs

To assess the effects of radiation on bronchial epithelium, BEAS 2B cells cultured as monolayers and human bronchial epithelium cultured as organ cultures were exposed to single doses of 0, 10 and 30 Gy. The lactate dehydrogenase in the supernatant of the BEAS 2B cells increased markedly 24 h after irradiation, whereas in the organ cultures only a minor increase was found after 48 h. The nucleosomes in the supernatant of the BEAS 2B cells showed a massive increase in response to irradiation, whereas in the organ cultures no change could be seen. The number of BEAS 2B cells was dramatically diminished after 96 h, whereas in the organ cultures a smaller decrease was observed no earlier than 21 days after irradiation. To assess the effects of brachytherapy in bronchial epithelium in vivo, brachytherapy with 30 Gy was performed in Goettinger minipigs, and histological sections of the bronchi were analyzed for morphological alterations and cell numbers. After 2 weeks, only slight cell damage was observable, and after 3 weeks, moderate morphological changes and decreased cell numbers were found. However, after 8 weeks, the epithelium had nearly regained its normal structure. We conclude that the bronchial epithelium has a remarkably high radioresistance and that organ cultures, but not monolayers of BEAS 2B cells, reflect the effects of radiation in vivo.     

Morphological evidence of basal keratinocyte migration during the re-epithelialization process

The regeneration of wounded stratified epithelium is accomplished via the migration of keratinocytes from the margins of the wound. However, the process of keratinocyte migration on the wound surface and the role of epithelial stem cells during re-epithelialization remain to be elucidated. Therefore, we administered BrdU to embryonic mice and generated epithelial defects on the buccal mucosa of these mice at two weeks after birth, using CO₂ laser irradiation, with which we removed the entire thickness of the epithelium. In the unwounded epithelium, cytokeratin 14, p63, and BrdU were localized within the basal layer of the epithelium, but the majority of cells within the regenerated epithelium were immunopositive for these proteins. PCNA-negative and BrdU-positive basal keratinocytes, which evidence a slow cell cycle, were localized solely within the basal layer of the unwound epithelium facing the tips of dermal papillae. After laser irradiation, these basal keratinocytes facing the tips of the papillae evidenced positive immunoreactivity for PCNA, in addition to BrdU. These results indicate that epithelial stem cells of oral mucosa may be localized in the basal layer of the epithelium facing the tips of dermal papillae, and may migrate laterally with other basal keratinocytes in response to external stimuli. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV^r-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.     

Buccal capsule development as a consideration for phylogenetic analysis of Rhabditida (Nemata)

 Bacterial feeding nematodes in the order Rhabditida including Zeldia punctata (Cephalobidae) and Caenorhabditis elegans (Rhabditidae) differ profoundly in the buccal capsule parts and associated cells. We carried out a range of tests to determine which buccal capsule parts and cells are evolutionarily homologous between the representative species of the two families. Tests included reconstruction of the buccal capsule and procorpus with transmission electron microscopy (TEM), nuclei position and morphology using 4,6-diamidino-2-phenylindole (DAPI) staining, and cell lineage using four dimensional (4D) microscopy. The lining of the buccal capsule of Z. punctata and additional Cephalobidae includes four sets of muscular radial cells, ma, mb, mc and md, in contrast to C. elegans and additional Rhabditidae, which has two sets of epithelial cells (e1, e3) and two sets of muscle cells (m1, m2). Cell lineage of a nematode closely related to Z. punctata, Cephalobus cubaensis, supports the hypothesis that in cephalobids the e1 and e3 cells become hypodermal cells or are programmed to die. Our findings contradict all previous hypotheses of buccal capsule homology, and suggest instead that ma and mb in Z. punctata are homologous to m1 and m2 in C. elegans respectively. We also hypothesize that ma and mb could be homologous to primary and secondary sets of stylet-protractor muscle cells in the plant parasitic Tylenchida. Received: 24 March 1998 / Accepted: 24 July 1998     

Patterns of epithelial expression of Fos protein suggest important role in the transition from viable to cornified cell during keratinization

An antibody directed against the DNA-binding region of c-fos was used to localize the distribution of cells positive for Fos protein in epithelial tissues. The antibody consistently bound to the nuclei of epithelial cells in the late stages of differentiation, just prior to cornification. The epidermis, palate, buccal mucosa, gingiva, tongue, forestomach and vagina in estrus all produced this type of labelling, suggesting a burst of expression immediately before cell death and cornification. The differentiating cells of the hair follicle, including the hair and inner root sheath, were also labelled. Non-keratinized tissues including junctional epithelium, embryonic epidermis and diestrus vaginal epithelium showed little or no Fos labelling. With the onset of keratinization at 18 days gestation or with induction of estrus in ovariectomized mice with estradiol benzoate, the epidermis and vagina expressed Fos protein in the manner typical for keratinized tissues. The Er/Er mutant epidermis, a tissue that is blocked in its ability to keratinize, overexpresses Fos with Fos-positive cells appearing in virtually every cell layer. Gel shift analysis demonstrates the presence of a functional AP-1 complex in epidermal extracts that is recognized by our antibody. Our data suggest that the expression of Fos is intricately related to epithelial cell differentiation, specifically in relation to the process of cornification and cell death.     

Apoptosis, cell proliferation and serotonin immunoreactivity in gut of Liza aurata from natural heavy metal polluted environments: preliminary observations

In the present paper, the effect of natural environment non-lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respectively, apoptosis and cell proliferation. In addition, the presence and distribution of the cell renewal regulator, serotonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were considered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intestinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and frequency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell proliferation, probably leading to morphological intestinal fold changes.     

Ontogeny of the digestive tract during larval development of yellowtail flounder: a light microscopic and mucous histochemical study

The histological development and mucous histochemistry of the alimentary tract in larval yellowtail flounder were studied using light microscopy. Samples were taken when the larvae were first offered food at 3 days post-hatch, then at 7, 10, 29, 36, and 46 days post-hatch, at which time they were metamorphosing. Regional partitioning of the digestive tract into the buccal cavity, pharynx, oesophagus, post-oesophageal swelling (PES), intestine, and rectum was complete by day 10. Goblet cells were present only in the buccal cavity, pharynx and intestine by day 7, but increased in number and distribution as development continued. By day 29, the posterior zone of the oesophagus had a marked increase in goblet cell density and mucosal folding. At the transition from oesophagus to PES/stomach stratified epithelium with goblet cells changed abruptly to a columnar epithelium with no goblet cells. Multicellular glands in the PES of 36-day larvae allowed it to be defined as a stomach. The distinct brush border of columnar epithelium and the presence of goblet cells characterize the intestine and rectum. All goblet cells throughout the digestive tract were strongly positive for acid mucins as was the luminal layer of the stratified epithelia lining the buccal cavity, pharynx and oesophagus. The PES/stomach epithelium stained weakly for neutral mucins. No mucin staining was associated with the gastric glandular epithelium. The brush borders of the intestine and rectum were strongly positive for combinations of neutral and acid mucins.     

Measurement and characterization of micronuclei in exfoliated human cells by fluorescence in situ hybridization with a centromeric probe

The micronucleus (MN) assay in human exfoliated cells has been widely used to detect the genotoxic effects of environmental mutagens, infectious agents and heriditary diseases. Substantial variability characterizes the MN frequencies reported by different research groups. One reason for this may be the restricted resolution power of the Feulgen-Fast-Green staining that is routinely used. Here we describe a new version of the MN assay that employs fluorescent propidium iodide staining along with fluorescence in situ hybridization (FISH) with a centromeric probe. Buccal and urothelial cells were collected from 5 healthy unexposed female volunteers and 55 000 cells analyzed for MN frequency and abnormal nuclear events. The Feulgen-Fast-Green and the new fluorescent staining produced very similar results. The frequency of MN in buccal cells was 0.145±0.118% and in urothelial cells 0.083±0.074%. No correlation was found between the frequencies of MN in the two types of exfoliated cells. FISH with a centrometric probe allowed MN containing whole chromosomes with a centromere to be differentiated from those containing only acentric fragments. The former appear as a result of chromosome lagging in mitosis, while those without a centromere are due to chromosome breakage. In urothelial cells 43% of MN were centromere-negative and in buccal cells — 44%. Fluorescent staining provided more accurate scoring of degenerative cells than standard Feulgen-Fast-Green staining. The combined frequency of pycnotic cells, “broken eggs” and cells with fragmented nuclei did not exceed 2%, while that of karyorrhexis and karyolysis together was as high as 21%. Significant interindividual variability was found in the frequency of cells with karyolysis and karyorrhexis. Thus, the new version of micronucleus assay allows for MN to be scored more precisely, the mechanism of MN formation to be determined and abnormal nuclear events to be readily identified in exfoliated human cells. It is therefore ideal for studying genotoxicity in human populations using exfoliated cells from the mouth, bladder and nose.     

Cellular apoptosis]

The interrelations between lymphocytes and intestinal epithelium cells after total X-ray irradiation (15 Gy) were investigated autoradiographically in CBA mice. The labelled lymphocytes injected in the irradiated animals migrated in intestinal epithelium, the label being present within vacuoles of the crypt cells and later in their nuclei. The trophic function of lymphocytes and the nature of the so-called apoptosis are discussed.