Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)‐induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability

Up to date, the nature of the sepsis‐induced vascular leakage is understood only partially, which limits pharmacological approaches for its management. Here we studied the protective effect of cAMP using endotoxin‐induced hyperpermeability as a model for barrier dysfunction observed in gram‐negative sepsis. We demonstrated that the alleviation of lipopolysaccharide (LPS)‐induced barrier compromise could be achieved by the specific activation of either protein kinase A (PKA) or Epac with cAMP analogs Bnz‐cAMP or O‐Me‐cAMP, respectively. We next studied the involvement of PKA substrates VASP and filamin1 in barrier maintenance and LPS‐induced barrier compromise. Depletion of both VASP and filamin1 with the specific siRNAs significantly exacerbated both the quiescent cells barrier and LPS‐induced barrier dysfunction, suggesting barrier‐protective role of these proteins. VASP depletion was associated with the more severe loss of ZO‐1 peripheral staining in response to LPS, whereas filamin1‐depleted cells reacted to LPS with more robust stress fiber induction and more profound changes in ZO‐1 and VE‐cadherin peripheral organization. Both VASP and filamin1 phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin1 depletion on the PKA‐dependent alleviation of LPS‐induced barrier compromise. We observed that Bnz‐cAMP ability to counteract LPS‐induced hyperpermeability was attenuated only by VASP, but not filamin1 depletion. Our data indicate that while PKA‐dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS‐compromised monolayers, filamin1 phosphorylation is unlikely to play a significant role in this process. J. Cell. Physiol. 221: 750–759, 2009. © 2009 Wiley‐Liss, Inc.     

NADPH oxidase controls EGF-induced proliferation via the ERK1/2-independent mechanism

Using HyPer, a ratiometric GFP-based biosensor, the dynamics of H2O2 in living cells has been studied. It was found that activation of the receptor of the epidermal growth factor (EGF) in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidase. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase should be involved in the process. However, apocynin failed to alter the kinetics of the EGF-stimulated ERK1/2 activation. It was concluded that NADPH oxidase and intracellular H2O2 are the important downstream targets of the EGF receptor, which mediate the proliferation response by mechanisms distinct from the activation of the classical ERK1/2 MAP-kinase pathway.     

BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells

Anoxic and metabolic stresses in large‐scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro‐apoptotic proteins and over‐expression of anti‐apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro‐apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc‐finger nuclease‐mediated gene disruption. Zinc‐finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale‐down systems under conditions that mimic growth in large‐scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX‐ and BAK‐deleted cells produce two‐ to fivefold more IgG than wild‐type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double‐knockout cultures achieve equal or higher cell densities than unmodified wild‐type cultures and reach viable cell densities relevant for large‐scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330–340. © 2009 Wiley Periodicals, Inc.     

Fast and precise protein tracking using repeated reversible photoactivation

Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we report 'protein rivers tracking' technique based on repeated identical rounds of photoactivation and subsequent images averaging, which results in dramatic increase of imaging resolution for fast protein movement events.     

Relation of the intensity of metabolism with the process of determination in embryonic cell

Relation between the level of the power metabolism and the degree of the determination of the embryos was investigated by comparison of the experimental material with theory. It was shown that the high level of metabolism promotes the change of the system to a differentiated state (i.e. leads to the limitation of tissue potency). The lowering of metabolism intensity is connected with a dedifferentiation of the tissue (or with expansion of it potency).     

Proteinaceous complexes from mitochondrial contact sites

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.     

The submolecular mechanism of the initial stage of the electron coupling in the water oxidizing center of higher plants

The results of quantum chemistry calculations of the total energy and proton position in tyrosine-histidine system in a water-oxidizing complex are presented. On the basis of these calculations, the submolecular mechanism of the initial stage of the electron coupling was proposed.     

Geldanamycin induces mitotic catastrophe and subsequent apoptosis in human glioma cells

Geldanamycin (GA) binds to heat shock protein 90 (Hsp90) and interferes with its function which is to protect various cellular proteins involved in signaling, growth control, and survival from ubiquitination and subsequent degradation by the proteasome. Recently, we demonstrated that GA inhibited migration of glioma cells in vitro associated with downregulation of hypoxia-inducible factor (HIF-1 alpha) and phosphorylation of focal adhesion kinase (FAK) (Zagzag et al., 2003, J Cell Physiol 196:394-402). Here, we have investigated the mechanisms through which GA treatment of the T98G glioma cell line induces apoptosis. We found that GA treatment induced cell death in a caspase-dependent manner through activation of caspase-3 and PARP cleavage together with release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria. Use of synchronized T98G cells showed that GA treatment of glioma cells during S-phase enhanced cytotoxicity followed by M-phase arrest, resulting in mitotic catastrophe. In addition, apoptosis was associated with the downregulation of the survival protein, phosphorylated Akt (pAkt), an important signaling protein in the PI3K pathway, that is overexpressed in many cancers including gliomas. Given that many glioma tumors show deregulation of the PI3K signaling pathway, either through loss of the tumor suppressor protein PTEN or overexpression of the growth factor EGFR, the ability to identify different subsets of patients using simple immunohistochemistry for the presence of absence of pAkt could enable selection of the appropriate kinase inhibitor, such as GA, for drug therapy. Based on our data presented here, GA or its analogs may have potential in the treatment of glioma.