Elena Stephanova

Cu/Zn superoxide dismutase in yeast mitochondria ^ a general phenomenon

Fermentative and respiratory yeast strains of genera Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula have been investigated for mitochondrial localization of Cu/Zn superoxide dismutase (SOD). Pure mitochondrial fractions were obtained and the specific activities of Cu/Zn and Mn SODs were measured in comparison with those in the corresponding cell-free extracts. The Cu/Zn SOD: Mn SOD ratio in mitochondria and crude extracts was calculated and was considered a specific characteristic of all tested strains. Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria. The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts.

Binding of sequences from the 5′- and 3′-nontranscribed spacers of the rat rDNA locus to the nucleolar matrix

Nucleolar matrix structures were obtained under different extraction conditions from highly purified isolated nucleoli. Their ultrastructural appearance, protein composition and capacity to bind rDNA preferentially were studied in a model binding system. A region spanning approximately 25 kb in the rat ribosomal gene locus was screened for DNA sites capable of specifically interacting with the proteins of the nucleolar matrix (MARs). Two such sites were identified: one is located on an EcoRV-KpnI fragment in the 5′-nontranscribed spacer region, between two repetitive elements and close to the transcription initiation site; the other MAR is on a PvuII-BamHI fragment located in the 3′-nontranscribed region, encompassing an element 85% homologous to a B2-sequence. The two MARs are located in regions rich in polypyrimidine/polypurine tracks and contain a few elements homologous to the consensus sequence for topoisomerase II. This indicates that the “attachment sites” for the ribosomal genes belong to the same class of sequences as the MARs attaching the chromosomal DNA to the nuclear matrix.

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-L-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-L-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.

Mitochondria are involved in stress response of A549 alveolar cells to halothane toxicity.

During inhalation anaesthesia lung epithelial cells are directly exposed to halogenated hydrocarbons such as halothane. Information about the effects of volatile anaesthetics on lung cells is rather limited although their noxious effect on the A549 alveolar cells has been shown recently. The present study indicated that halothane decreases cell viability, impairs DNA integrity and provokes stress-induced apoptosis in A549 cells when applied at clinically relevant concentrations. Data obtained clearly demonstrated intensive expression of anti-apoptotic Bcl-2 protein during treatment with all tested concentrations. In post-treatment periods the increased DNA injury was accompanied by reduction of Bcl-2 expression. We concluded that the in vitro effect of halothane on lung cells involved alteration in the expression of proteins of the mitochondrial apoptotic pathway.

Influence of Volatile Anaesthetics on Lung Cells and Lung Surfactant

ABSTRACT Volatile anaesthetics as halothane, enflurane, isoflurane, sevoflurane and desfluane have significant role in surgery. Anaesthesia could affect breath holding and might cause a dose-dependent depression in respiration. Lung epithelial cells are the first barrier against inhaled agents. Among them the pneumocytes type II are the main producers of pulmonary surfactant. Damage of lung surfactant (LS) is one of the possible reasons for the respiratory complications. In contrast to studies, concerning the influence of general anaesthetics on lung surfactant functionality, there is still poor information about the influence, based on the alveolar cell cultures. In this review we focus our attention on the data indicating the harmful effect of anaesthetics and mainly of halothane on the properties of lung cells, biosynthesis, secretion and functionality of LS components, using an appropriate cell culture model system and we discuss as far as possible the mechanism underlying the halothane toxicity.

Halothane-induced alterations in cellular structure and proliferation of A549 cells

Genotoxicity, cytotoxicity or teratogenicity are among the well-known detrimental effects of the volatile anaesthetics. The aim of the present work was to study the structural changes, proliferative activity and the possibility of alveolar A549 cells to recover after in vitro exposure to halothane at 1.5 and 2.1mM concentrations. Our data indicated significant reduction of viability, suppression of mitotic activity more than 60%, and that these alterations were accompanied by disturbances of nuclear and nucleolar structures. The most prominent negative effect was the destruction of the lamellar bodies, the main storage organelles of pulmonary surfactant, substantial for the lung physiology. In conclusion, halothane applied at clinically relevant concentrations exerts genotoxic and cytotoxic effect on the alveolar cells in vitro, most likely as a consequence of stress-induced apoptosis, thus modulating the respiratory function.

Halothane affects focal adhesion proteins in the A 549 cells.

Halothane is a volatile anaesthetic, which is known to induce alterations in cellular plasma membranes, modulating the physical state of the membrane lipids and/or interacting directly with membrane-bound proteins, such as integrin receptors. Integrin-mediated cell adhesion is a general property of eukaryotic cells, which is closely related to cell viability. Our previous investigations showed that halothane is toxic for A 549 lung carcinoma cells when applied at physiologically relevant concentrations and causes inhibition of adhesion to collagen IV.The present study is focused on the mechanisms underlying halothane toxicity. Our results imply that physiologically relevant concentrations of halothane disrupt focal adhesion contacts in A 549 cells, which is accompanied with suppression of focal adhesion kinase activity and paxillin phosphorylation, and not with proteolytic changes or inhibition of vinculin and paxillin expression.We suggest that at least one of the toxic effects of halothane is due to a decreased phosphorylation of the focal contact proteins.

Gamma-Glutamyl Transpeptidase Activity in Human Fetal Lung-Derived Cells

ABSTRACT Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) - a membrane-associated enzyme is a main regulator of glutathione (GSH) levels. The enzyme is not only the inhibitor of apoptosis induced by oxidative stress, but it is engaged in anti-tumor drug cell resistance and is considered as a marker for neoplastic changes, cell differentiation and aging. The surface and total activities of GGT in unfixed, fixed with para-formaldehyde vapors P cells (human fetal lung-derived cells) and cells lyzates were measured and compared using the γ-Glu-4-nitroanilide substrate. Our results showed that the surface activity was higher than the total, which might be due to the desegregation of the enzyme molecules upon mechanical cell homogenization and at the same time, after cell fixation in para-formaldehyde vapors it was decreased twice. Thus, methods including homogenization and/or aldehyde fixation are not suitable for the study of GGT activity. In addition, visualization of GGT activity was tested using the fluorogenic substrate γ-Glu-4-hydrazido-N-hexyl-1,8- naphthalimide (γ-Glu-HHNI). Our results pointed out that GGT could not be visualized in fixed P (normal diploid) cells using this substrate probably as a result of the lower reactivity of the enzyme towards hydrazide substrates in comparison to amide substrates. On the other hand, GGT activity was well demonstrated with the same substrate in SK-MES-1 cells (human squamous cell carcinoma). Consequently, the novel fluorogenic substrate γ-Glu-HHNI could be used for diagnostic purposes on the basis of not-detectable/detectable GGT activity in normal and pathologically altered lung epithelial cells respectively.