To be remembered: Bogumił Leon Kmieć (1944-2004) p. 172
ABSTRACTS
Quercetin suppresses heat shock-induced nuclear translocation of Hsp72
Joanna Jakubowicz-Gil, Bożena Pawlikowska-Pawlęga, Tomasz Piersiak, Jarosław Pawelec and Antoni Gawron
Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Lublin, Poland
Abstract: The effect of querectin and heat shock on the Hsp72 level and distribution in HeLa cells was studied by Western blotting, indirect immunofluorescence and immunogold electron microscopy. In control cells and after quercetin treatment, Hsp72 was located both in the cytoplasm and in the nucleus in comparable amounts. After hyperthermia, the level of nuclear Hsp72 raised dramatically. Expression of Hsp72 in cytoplasm was also higher but not to such extent as that observed in the nucleus. Preincubation of heated cells with quercetin inhibited strong Hsp72 expression observed after hyperthermia and changed the intracellular Hsp72 distribution. The cytoplsmic level of protein exceeded the nuclear one, especially around the nucleus, where the coat of Hsp72 was noticed. Observations indicating that quercetin was present around and in the nuclear envelope suggested an involvement of this drug in the inhibition of nuclear translocation. Our results indicate that pro-apoptotic activity of quercetin may be correlated not only with the inhibition of Hsp72 expression but also with suppression of its migration to the nucleus.
Author’s e-mail: jjgil@biotop.umcs.lublin.pl
J. Saczko1, A. Chwiłkowska1, M. Drąg-Zalesińska2, T. Wysocka, M. Ługowski1, J. Kulbacka1 and T. Banaś1
1Department of Medical Biochemistry and 2Department of Histology and Embryology, Medical University, Wrocław, Poland
Abstract: Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegröm Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 μg/ml and 30 μg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 μg/ml and 30 μg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.
Author’s e-mail: michal@bioch.am.wroc.pl
Expression of natural antimicrobial peptide b -defensin-2 and Langerhans cell accumulation in epidermis from human non-healing leg ulcers
Hanna Galkowska1 , Waldemar L Olszewski1,3 and Urszula Wojewodzka2
1Department of Surgery and Transplantation and 2Laboratory of Cell Ultrastructure, Medical Research Center, Polish Academy of Sciences; 3Central Clinical Hospital, Ministry of Internal Affairs, Warsaw, Poland
Abstract: Chronic wounds like venous calf and diabetic foot ulcers are frequently contaminated and colonized by bacteria and it remains unclear whether there is sufficient expression of defensins and recruitment of epidermal Langerhans cells in the margin of ulcer compared to normal skin. The aim of this study was to examine immunohistochemically the expression of b -defensin-2 (hBD2), GM-CSF, VEGF growth factors and accumulation of CD1a+ Langerhans cells (LC) in epidermis from chronic skin ulcers and to compare it to normal skin from the corresponding areas. Studies were carried out in 10 patients with diabetic foot, 10 patients with varicous ulcers of the calf and 10 patients undergoing orthopedic surgery (normal skin for control). Biopsy specimens were immunostained using specific primary antibodies, LSAB+ kit based on biotin- avidin- peroxidase complex technique and DAB chromogen. Results were expressed as a mean staining intensity. Statistical analysis of staining showed significantly higher staining of hBD2 in both normal and ulcerated epidermis from foot sole skin compared to calf skin (normal and ulcerated, p<0.05). Chronic ulcers showed the same expression of hBD2 as normal skin. There was significantly lower accumulation of CD1a+ LC in normal epidermis from foot sole skin compared to normal calf skin (p<0.05). Accumulation of CD1a+ LC and GM-CSF upregulation at the border area of diabetic foot ulcer and reduction of LC concentration at the margin of venous calf ulcer compared to normal skin were observed. It seems that normal calf and sole epidermis is, unlike in the mechanisms of innate immunity, influenced by the different keratinocyte turnover and bacterial flora colonizing these regions. Insufficient upregulation of hBD2 in both diabetic foot and venous calf ulcers may suggest the pathological role of this protein in the chronicity of ulcers.
Author’s e-mail: hgalk@cmdik.pan.pl
Immunohistochemical detection of PPARγ receptors in the human pituitary adenomas: correlation with PCNA
Katarzyna Winczyk and Marek Pawlikowski
Department of Experimental Endocrinology and Hormone Diagnostics, Institute of Endocrinology, Medical University, Łódź, Poland
Abstract: The occurrence of peroxisome proliferator-activated receptors gamma (PPARγ) was investigated in 51 human pituitary adenomas and in 6 non-tumoral human pituitary tissue samples was investigated. Moreover, the correlation between PPARγ and the proliferating cells nuclear antigen (PCNA) – immunocytochemical proliferation marker was evaluated. The receptors and PCNA were detected by immunohistochemical methods using the polyclonal anti-PPARγ and the monoclonal anti-PCNA antibodies, respectively. PPARγ were found in all examined tissues. The mean percentage of cells with positive nuclear reaction was 3-fold higher in pituitary adenomas in comparison with non-tumoral pituitary tissues. The strongest expression of PPARγ was observed in somatotropinomas. Besides the nuclear reaction, which is typical for PPARγ, positive immunostaining was also observed in the cytoplasm. It was clearly stronger in pituitary adenomas than in non-tumoral pituitary tissues. A slight, statistically insignificant tendency towards negative correlation between PPARγ and PCNA was found in somatotropinomas, prolactinomas, corticotropinomas and gonadotropinomas. On the other hand, in null cell adenomas and “silent” corticotropinomas, a strong positve correlation between the expression of PPARγ and PCNA was observed. The strong expression of PPARγ in human pituitary adenomas and its possible involvement in control of cell proliferation in these tumors give a good reason for the attempts of their treatment with PPARγ ligands.
Author’s e-mail: kwinczyk@os1.kappa.pl
Effects of excess vitamin B6 intake on cerebral cortex neurons in rat: an ultrastructural study
Ramazan Demir1 , Goksemin Acar2, Gamze Tanriover1, Yasemin Seval1, Umit Ali Kayisli1 and Aysel Agar3
1Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya;
2Department of Neurology, Faculty of Medicine, Pamukkale University, Denizli;
3Deaprtment of Physiology, Faculty of Medicine, Akdeniz University, Antalya; Turkey.
Abstract: The aim of this study was to investigate whether excess of vitamin B6 leads to ultrastructural changes in cerebral cortex of forty-eight healthy albino rats which were included in the study. Saline solution was injected into to the control groups (CG-10, n=12 for 10 days; CG-15, n=12 for 15 days; CG-20, n=12 for 20 days). The three experimental groups (EG-10, n=12; EG-15, n=12; EG-20, n=12) were treated with 5 mg/kg vitamin B6 daily for 10 days (EG-10), 15 days (EG-15) and 20 days (EG-20). Brain tissues were prepared by glutaraldehyde-osmium tetroxide double fixation for ultrastructural analysis. No significant changes were observed in the control groups. The ultrastructural analysis revealed that the numbers of damaged mitochondria, lipofuscin granules and vacuoles were significantly higher in all the experimental groups than in the control groups (p<0.05). However, synaptic density was significantly decreased in the experimental groups as compared to the control groups (p<0.05). The results suggest that the excess of vitamin B6 intake causes damage to the cerebral cortex due to cellular intoxication and decreased synaptic density. Thus, careful attention should be paid to the time and dose of vitamin B6 recommended for patients who are supplemented with this vitamin.
Author’s e-mail: rdemir@akdeniz.edu.pl
Concetta Calabrò1, Maria Pia Albanese1, Silvestro Martella1, Patrizia Licata2, Eugenia Rita Lauriano1, Clara Bertuccio1 and Aurelio Licata1
1Department of Animal Biology and Marine Ecology, Faculty of Science, and 2Department of Veterinary Public Health, Section of Veterinary Pharmacology and Toxicology, University of Messina, Italy
Abstract: Glycosaminoglycans and NO synthase probably regulate mucous cell secretion in the skin of Tapes philippinarum. We have demonstrated the presence of “protein” cells, “glycogen” cells, “phenol” cells and five types of mucous cells, with different chemical composition of the mucus in the mantle epithelium of T. philippinarum. The foot epithelium contained “protein” cells and two types of mucous cells. Using biotinylated lectins, in the mantle and foot epithelia we have shown specific sites for the following oligosaccharides: α-D-glucose, α-D-mannose, α-L-fucose, a-D-1,3-N-acetyl-galactosamine and α-N-acetyl-glucosamine. nNOS immunoreactivity in the intraepithelial and intradermal cells and in the mucocytes suggested a regulatory role of NO in mucus secretion, as demonstrated also in other invertebrates.
Author’s e-mail: alicata@unime.it
Full textEffect of aminoguanidine and albendazole on inducible nitric oxide synthase (iNOS) activity in T. spiralis-infected mice muscles
Jan Żeromski1, Krystyna Boczoń2, Elżbieta Wandurska-Nowak2 and Iwona Mozer-Lisewska3
1Department of Clinical Immunology, 2Department of Biology and Medical Parasitology and 3Department of Child Infectious Diseases, University of Medical Sciences, Poznań, Poland
Abstract: The aim of this study was to provide evidence for the expression of iNOS in the cells of inflammatory infiltrates around larvae in skeletal muscles of T. spiralis infected mice. The BALB/c mice (n=8) divided into subgroups, received either aminoguanidine (AMG) - a specific iNOS inhibitor or albendazole (ALB)- an antiparasitic drug of choice in trichinellosis treatment. Control animals (n=2 in each subgroup) were either uninfected and treated or uninfected and untreated. Frozen sections of hind leg muscles from mice sacrificed at various time intervals after infection were cut and subjected to immunohistochemistry, using monoclonal anti-iNOS antibody. The ALB-treated mice revealed stronger iNOS staining in the infiltrating cells around larvae than the infected and untreated animals. On the contrary, in the AMG-treated animals, the infiltrating cells did not show any specific iNOS reaction. These data confirm the specificity of iNOS staining in the cellular infiltrates around T. spiralis larvae and shed some light on the role of nitric oxide during ALB treatment in experimental trichinellosis.
Author’s e-mail: kboczon@amp.edu.pl
Magdalena Mosiołek1, Paweł Pasierbek2, Janusz Malarz3, Maria Moś4 and Andrzej J. Joachimiak1
1Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Kraków, Poland;
2Department of Cell Biology and Genetics, Institute of Botany, University of Vienna, Vienna, Austria;
3Department of Phytochemistry, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland;
4Department of Plant Breeding and Seed Science, Agricultural University, Kraków, Poland
Abstract: Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Stearn roots. It was shown, that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.
Author’s e-mail: a.joachimiak@iphils.uj.edu.pl
of children with newly diagnosed lymphomas
Włodzimierz Łuczyński1, Oksana Kovalchuk4, Maryna Krawczuk-Rybak1, Małgorzata Mitura-Lesiuk2, Iwona Malinowska3, Lech Chyczewski4, Jerzy Kowalczyk2 and Michał Matysiak3
1Department of Pediatric Hematology and Oncology, Medical University, Białystok;
2Department of Pediatric Hematology and Oncology, Medical University, Lublin;
3Department of Pediatric Hematology and Oncology, Medical University, Warszawa;
4Department of Clinical Molecular Biology, Medical University, Białystok, Poland
Abstract: The complex interactions between cancer and host cells are far from being fully elucidated. Assessment of Th1/Th2/Th3/Tr1 balance is an interesting approach to explain immunological disturbances in lymphomas. The aim of our study was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkin lymphomas. CD4+ and CD8+ cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-g , IL-10, IL-4, TGF-b ) were determined by real-time PCR technique. Comparing to the control group, we found lower expression of mRNA for IFN-gamma in CD4+ cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms of impaired immunity in these patients.
Author’s e-mail: vlodek@amb.edu.pl
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