Monosodium glutamate (MSG), the sodium salt of glutamate, is commonly used as a flavor enhancer in modern nutrition. Recent studies have shown the existence of glutamate receptors on lymphocytes, thymocytes and thymic stromal cells. In this study, we evaluated the in vitro effect of different MSG concentrations on rat thymocyte apoptosis and expression of two apoptosis-related proteins, Bcl-2 and Bax. Rat thymocytes, obtained from male Wistar rats, were exposed to increasing concentrations of MSG (ranging from 1 mM to 100 mM) for 24 h. Apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit and cells were analyzed using a flow cytometer. Expression of Bcl-2 and Bax proteins were determined with flow cytometry using respective monoclonal antibodies. Exposure to MSG resulted in a dose-dependent decrease in cell survival (as determined by trypan blue exclusion method). Annexin VFITC/ PI also confirmed that MSG increased, in a dose-dependent manner, apoptotic cell death in rat thymocyte cultures. MSG treatment induced downregulation of Bcl-2 protein, while Bax protein levels were not significantly changed. Our data showed that MSG significantly modulates thymocyte apoptosis rate in cultures. The temporal profile of Bcl-2 and Bax expression after MSG treatment suggests that downregulation of Bcl-2 protein and the resulting change of Bcl-2/Bax protein ratio may be an important event in thymocyte apoptosis triggered by MSG.

• MeSH
• Apoptosis genetics   drug effects   radiation effects   MeSH
• Financing, Organized utilization   MeSH
• Sodium Glutamate adverse effects   MeSH
• Antibodies, Monoclonal diagnostic use   MeSH
• Rats, Wistar MeSH
• bcl-2-Associated X Protein genetics   immunology   adverse effects   MeSH
• Flow Cytometry methods   utilization   MeSH
• T-Lymphocytes MeSH
• Thymus Gland enzymology   physiology   MeSH

Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.

• MeSH
• Calorimetry, Differential Scanning MeSH
• Phosphorylcholine analogs & derivatives   metabolism   MeSH
• Humans MeSH
• Carbon-13 Magnetic Resonance Spectroscopy MeSH
• Mitochondrial Membranes metabolism   MeSH
• Oxidation-Reduction MeSH
• Permeability MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Proton Magnetic Resonance Spectroscopy MeSH
• Unilamellar Liposomes MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The pro-apoptotic proteins Bax and Bak are essential for executing programmed cell death (apoptosis), yet the mechanism of their activation is not properly understood at the structural level. For the first time in cell death research, we calculated intra-protein charge transfer in order to study the structural alterations and their functional consequences during Bax activation. Using an electronegativity equalization model, we investigated the changes in the Bax charge profile upon activation by a functional peptide of its natural activator protein, Bim. We found that charge reorganizations upon activator binding mediate the exposure of the functional sites of Bax, rendering Bax active. The affinity of the Bax C-domain for its binding groove is decreased due to the Arg94-mediated abrogation of the Ser184-Asp98 interaction. We further identified a network of charge reorganizations that confirms previous speculations of allosteric sensing, whereby the activation information is conveyed from the activation site, through the hydrophobic core of Bax, to the well-distanced functional sites of Bax. The network was mediated by a hub of three residues on helix 5 of the hydrophobic core of Bax. Sequence and structural alignment revealed that this hub was conserved in the Bak amino acid sequence, and in the 3D structure of folded Bak. Our results suggest that allostery mediated by charge transfer is responsible for the activation of both Bax and Bak, and that this might be a prototypical mechanism for a fast activation of proteins during signal transduction. Our method can be applied to any protein or protein complex in order to map the progress of allosteric changes through the proteins' structure.

• MeSH
• Allosteric Regulation MeSH
• Apoptosis physiology   MeSH
• Models, Biological MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Computer Simulation MeSH
• bcl-2 Homologous Antagonist-Killer Protein chemistry   genetics   metabolism   MeSH
• bcl-2-Associated X Protein chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Static Electricity MeSH
• Computational Biology MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Validation Study MeSH

• MeSH
• Cataract Extraction MeSH
• Genes, bcl-2 MeSH
• Lens Implantation, Intraocular MeSH
• Presbyopia MeSH
• bcl-2-Associated X Protein MeSH
• Refractive Surgical Procedures MeSH
• Retinal Pigments MeSH
• Publication type
• Collected Work MeSH

• Conspectus
• Ortopedie. Chirurgie. Oftalmologie
• NML Fields
• oftalmologie

The aim of our in vitro studies was to understand the role of leptin in controlling proliferation, apoptosis, and protein kinase A (PKA) in human ovarian cells. We analyzed the in vitro effects of leptin (0, 1, 10 or 100 ng/ml) on the accumulation of proliferation-related peptides (PCNA, cyclin B1), apoptosis-associated peptide (Bax) and the intracellular signaling molecule PKA in cultured human granulosa cells using immunocytochemistry and Western immunoblotting. It was observed that leptin stimulated in a dose-dependent manner the accumulation of PCNA (at doses 1-100 ng/ml), cyclin B1 (at doses 10 or 100 ng/ml), Bax (at doses 10 or 100 ng/ml) and PKA (at doses 1-100 ng/ml) in cultured human ovarian cells. These observations suggest the ability of leptin to control directly human ovarian cell functions: proliferation, apoptosis, and intracellular messenger PKA.

• MeSH
• Apoptosis MeSH
• Cell Cycle MeSH
• Cyclin B metabolism   MeSH
• Adult MeSH
• Financing, Organized MeSH
• Granulosa Cells enzymology   immunology   pathology   MeSH
• Immunohistochemistry MeSH
• Cells, Cultured MeSH
• Leptin metabolism   MeSH
• Humans MeSH
• Cell Proliferation MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Signal Transduction MeSH
• Blotting, Western MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH

Etoposide is a well-known and widely used anticancer drug that displays several side effects. In addition, tumors often acquire resistance to this drug. Our aim is to develop a combination therapy that would augment toxicity of etoposide in malignant cells. Based on literature and our experiments, we selected mifepristone (RU486) as a potential supporting molecule that is able to enhance etoposide toxicity against cancer cells. All experiments were performed with Hep G2 cells, a well-known and described human hepatocellular carcinoma cell line. By using xCELLigence system, we demonstrated that mifepristone enhances toxicity of etoposide in a dose dependent manner with concomitant caspase-3 activity. We evaluated upregulation of Bax because mifepristone was demonstrated to modulate proapoptotic Bax protein expression. Our data show only weak and not statistically significant increase of Bax expression. On the other hand, we show that mifepristone increases etoposide toxicity via inhibition of ABC transporters, coupled with significant increase of intracellular etoposide concentration. In conclusion, we demonstrate that mifepristone has a synergistic effect with etoposide treatment in the Hep G2 cells and that the effect is related to ABC transporters inhibition.

• MeSH
• ATP Binding Cassette Transporter, Subfamily G, Member 2 antagonists & inhibitors   MeSH
• Biological Transport drug effects   MeSH
• Hep G2 Cells MeSH
• K562 Cells MeSH
• Etoposide pharmacology   MeSH
• Antineoplastic Agents, Phytogenic pharmacology   MeSH
• Caspase 3 metabolism   MeSH
• Humans MeSH
• Mifepristone pharmacology   MeSH
• Neoplasm Proteins antagonists & inhibitors   MeSH
• ATP Binding Cassette Transporter, Subfamily B antagonists & inhibitors   MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Drug Synergism MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The maintenance of tissue homeostasis and highly balanced counteraction of cellular proliferation and apoptosis are essential for tissue integrity. In our study, we evaluated the expression of apoptosis- -regulating proteins Bcl-2, Bax and PARP, and executive apoptotic enzyme caspase-3 in normal, atrophic, hyperplastic and cancerous endometrium. Endometrial samples were obtained from patients who underwent curettage, hysteroresection or hysterectomy. The protein levels were quantified by immunoblotting. We observed a higher level of important apoptotic enzyme pro-caspase-3 and its active form in hyperplastic and cancerous endometrium, when compared to normal endometrium. The value of Bcl-2/Bax ratio, which reflects cellular resistance to apoptosis, was determined as > 1 for cancerous, normal, and atrophic endometrium. Thus, the effort to eliminate pre-neoplastic and neoplastic cells by apoptosis indicated by high pro-caspase-3 and caspase-3 levels seems to be overcome by a greater proliferative adjustment suggested by higher Bcl-2/Bax ratios in the samples examined. The PARP levels did not vary significantly among the groups. The levels of all examined proteins were significantly lower in atrophic endometrium. Our results suggest that pre-neoplastic and neoplastic states of human endometrium are not influenced simply by changes in apoptosis, but may also be affected by cellular proliferation. A high Bcl-2/Bax ratio as observed in cancerous endometrium can point to deregulation of apoptotic programmes. Thus, the onset and progression of endometrial malignancy could be linked to increased cellular proliferation with defects in apoptotic control.

• MeSH
• Apoptosis physiology   drug effects   MeSH
• Adult MeSH
• Endometrium metabolism   pathology   MeSH
• Endometrial Hyperplasia metabolism   pathology   MeSH
• Immunohistochemistry MeSH
• Carcinoma metabolism   pathology   MeSH
• Caspase 3 metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Endometrial Neoplasms metabolism   pathology   MeSH
• Cell Proliferation MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Apoptosis Regulatory Proteins metabolism   MeSH
• Proto-Oncogene Proteins c-bcl-2 metabolism   MeSH
• Aged MeSH
• Blotting, Western MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

Prospektivní studie zabývající se predikcí výskytu recidiv a progrese povrchových NMM za základě stanovení expresních profilů kandidátních genů PAX5, HSP60, BCL2 a BAX v tkáni karcinomu močového měchýře. Dalším cílem je rozpracovat metodiku semikvantitativního způsobu hodnocení imunohistochemického vyšetření exprese PAX5, zjistit korelaci s mírou exprese zjišťovanou pomocí RT-PCR a zavést imunohistochemii PAX5 do klinické praxe. Dalším cílem je zjistit zda imunohistochemické vyšetření exprese p53, Ki-67a PAX5 ve vzorcích nenádorové sliznice, odebírané při transuretrální resekci povrchového NMM, přináší prognostickou informaci stran recidivy či progrese onemocnění. Studie přinese návrh použití prognostických markerů při sledování a plánování léčby pacientů s povrchovým nádorem měchýře, což bude mít přínos pro kvalitu pacientova života i ekonomický.; The prospective study dealing with the recurrence and progression of superficial bladder cancer prediction on the basis of expression profiles of candidate genes PAX5, HSP60, BCL2 and BAX in tumour biopsies. Another aim of the study is to develop methodology of semiquantitative immunohistochemical examination of PAX5 experession, to find out the correlation with the expression detected by RT-PCR and put PAX5 immunohistochemistry into clinical practice. Next aim of the study is to find out if the immunohistochemical examination of p53, Ki-67 and PAX5 expression in non-tumour mucosa samples taken during the superficial bladder carcinoma transurethral resection brings any prognostic information. The study will bring a suggestion of using the prognostic markers when following patients with superficial bladder carcinoma and when planning the treatment, which will be benefical for them.

• MeSH
• PAX5 Transcription Factor analysis   MeSH
• Chaperonin 60 analysis   MeSH
• Gene Expression immunology   MeSH
• Immunohistochemistry methods   utilization   MeSH
• Urinary Bladder Neoplasms MeSH
• Reverse Transcriptase Polymerase Chain Reaction methods   utilization   MeSH
• Prognosis MeSH
• bcl-2-Associated X Protein analysis   MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• urologie
• onkologie
• biologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

BACKGROUND: Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. RESULTS: Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. CONCLUSION: In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Apoptosis genetics   MeSH
• Cell Line MeSH
• CD4-Positive T-Lymphocytes metabolism   MeSH
• DNA Fragmentation MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• HIV Infections genetics   metabolism   MeSH
• HIV-1 genetics   metabolism   MeSH
• HIV Protease genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Promoter Regions, Genetic genetics   MeSH
• bcl-2-Associated X Protein genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.

• MeSH
• Apoptosis * genetics   MeSH
• Respiration MeSH
• Humans MeSH
• Mitochondria genetics   metabolism   MeSH
• Mice MeSH
• bcl-2 Homologous Antagonist-Killer Protein * genetics   metabolism   MeSH
• bcl-2-Associated X Protein genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-bcl-2 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH