Federico Cusinato

Lipid interaction of diphtheria toxin and mutants with altered fragment B

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.

Cardiac glycoside ouabain induces autophagic cell death in non-small cell lung cancer cells via a JNK-dependent decrease of Bcl-2.

Cardiac glycosides are Na/K-ATPase inhibitors, clinically used for congestive heart failure and cardiac arrhythmias. Epidemiological studies have reported that patients on cardiac glycosides treatment are protected from some types of cancers. This evidence together with the demonstration that cardiac glycosides show selective cytotoxicity against cancer cells has raised new interest on the anticancer properties of these drugs. This study examines the mechanism involved in the anticancer effect of ouabain in non-small cell lung cancer cells lines (A549 and H1975). Ouabain inhibited cell proliferation and induced cell death in a concentration-dependent manner. Cell death was caspase-independent and showed classical patterns of autophagic cell death: conversion of LC3-I to LC3-II, increase of LC3 puncta and increase of autophagic flux. Moreover, cell death was completely blocked by the class III phosphatidylinositol-3 kinase inhibitor 3-methyladenine. Here we show that ouabain caused the reduction of Bcl-2 protein levels, with no change in the expression of the autophagic protein Beclin 1. Early signalling events of ouabain exposure were ERK1/2 and JNK activation, however only JNK inhibition with SP600125 or JNK knockdown by shRNA were able to prevent Bcl-2 decrease, conversion of LC3-I to LC3-II and cell death. We propose that JNK activation by ouabain leads to a decrease of Bcl-2 levels, resulting in disruption of the inhibitory interaction of Bcl-2 with Beclin 1, that promotes autophagy. These findings indicate that pharmacological modulation of autophagy by cardiac glycosides could be exploited for anticancer therapy.

Identification and purification of calpactins from cardiac muscle and their effect on Na+Ca2+ exchange activity

Calpactins were purified from bovine cardiac muscle by a slightly modified Glenney et al. procedure (J. Cell. Biol. 104, 503-511, 1987). Two major proteins (apparent MW of 36 and 68 kDa) able to bind phospholipids in a Ca2(+)-dependent manner were identified. These proteins completely reversed the inhibition of Na+/Ca2+ exchange activity of cardiac sarcolemmal vesicles consequent to EGTA-treatment. A modulation of cardiac Na+/Ca2+ exchange activity by calpactins is suggested.

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids.

The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.

Antiapoptotic and Proliferative Effects of Low Concentrations of 7β-Hydroxycholesterol in Human Endothelial Cells via ERK Activation

The atherogenic potential of oxidized low-density lipoproteins (oxLDL) has been correlated to their 7β-hydroxycholesterol (7βOHC) content; oxLDLs have a dual effect on endothelial cell viability, inducing apoptosis or proliferation depending on the concentration. Considering that 7βOHC is apoptotic for endothelial cells at concentrations ≧20 µg/ml, a study on the effect of lower concentrations of 7βOHC on human umbilical vein endothelial cells (HUVECs) was undertaken. 7βOHC (1–10 µg/ml) increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction of growth-factor-deprived HUVECs. This effect was due to the increased cell proliferation, determined by [3H]thymidine incorporation, and the reduction of apoptosis, revealed by the decreased caspase-3 activation and annexin V staining. 7βOHC also protected against staurosporine apoptosis. 7βOHC induced an increase in intracellular ROS antagonized by N-acetylcysteine; however, HUVECs treatment with the antioxidant did not inhibit the effects of 7βOHC. 7βOHC produced an increase in extracellular signal-regulated kinase (ERK) phosphorylation that was blocked by inhibitors of store-operated calcium entry 2-aminoethoxydiphenyl borate and gadolinium. MEK inhibition with PD98059 or U0126 as well as store-operated calcium entry inhibition antagonized the effect of 7βOHC. The results suggest that 7βOHC promotes HUVECs survival and proliferation by a mechanism independent of ROS production and involving calcium-dependent activation of ERK.

Temperature dependence of Na+/Ca2+ exchange activity in beef-heart sarcolemmal vesicles and proteoliposomes.

Temperature dependence of Na+/Ca2+ exchange activity was studied in beef cardiac sarcolemmal vesicles in the absence and presence of the inhibitor amiloride and in proteoliposomes reconstituted with different lipid mixtures. Arrhenius plots for Na+/Ca2+ exchange activity in both control and amiloride-treated vesicles revealed an apparent energy of activation of 9665 +/- 585 (SE, n = 4) cal/mol, corresponding to a temperature coefficient (Q10) value of 1.70 +/- 0.05 (SE, n = 4) over the range 25-37 degrees C. When Na+/Ca2+ exchange was reconstituted into phosphatidylcholine (PC):phosphatidylserine (PS) (52:48, mol/mol), PC:PS:cholesterol (25:39:36, mol/mol), and PC:PS:distearoylphosphatidylcholine (DSPC) (31:48:21, mol/mol) proteoliposomes, the highest activity was found in PC:PS:cholesterol proteoliposomes. Arrhenius plots of Na+/Ca2+ exchange activity exhibited breakpoints at 23 degrees C (PC:PS), 33 degrees C (PC:PS:cholesterol), and 23 degrees C (PC:PS:DSPC). The increase in the thermotropic transition temperature with cholesterol could result from the condensing effect of this sterol, whereas the breaks observed with PC:PS and PC:PS:DSPC could be caused by a non-lipid-mediated membrane protein conformational change. These results indicate that the lipid microenvironment around the Na+/Ca2+ exchanger and the nature of the specific lipid-protein interactions influence the activity of this antiporter. Further evidence supporting the hypothesis that cholesterol behaves as a specific positive effector for the exchanger is also given.

Cholesterylphosphoserine as inhibitor of cell adhesion and actin polymerization in human T cells.

To further investigate the immunosuppressive activity of cholesterylphosphoserine (CPHS), we examined a variety of human T cell responses including proliferation, adhesion and cytoskeletal organization. The CPHS-induced inhibition of T cell response is greater in the integrin-dependent mixed lymphocyte reaction than in the integrin-independent proliferation elicited by anti-TCR-CD3 or anti-CD28 antibodies in the presence of tetradecanoylphorbol acetate. Consistently, CPHS inhibits the homotypic T cell adhesion involving the integrin alphaLbeta2 (LFA-1) and the cell adhesion to fibronectin and rVCAM-1 involving the integrins of the beta1 family. Since CPHS does not change integrin expression but inhibits post-receptor events such as cell spreading and pseudopodal projections, it seems likely that the site of CPHS influence is distal to the adhesion receptors. In agreement, the steroid prevents the reorganization of actin cytoskeleton occurring when T cells are allowed to spread on immobilized anti-CD3 in the absence of integrin activation. We suggest that CPHS acts on the metabolic pathway in which signals from integrin and growth factor receptors converge to induce the reorganization of the actin cytoskeleton. Selectivity in the action of CPHS is indicated by its ineffectiveness in the integrin-mediated adhesion of the monocytic cell line U-937 to fibronectin.

7β-hydroxycholesterol is antiapoptotic and induces proliferation in human endothelialcells by a ROS-independent ERK-dependent mechanism

Evaluation of antibiotic prescribing in upper respiratory tract infections R Vaishnav, W Al-Naeem, F Al-Omrani Sultan Qaboos University, Muscat, Oman The purpose of this study was to evaluate trends in antibiotic prescribing at Family and Community Health Clinics. Males, females and children who attended for the treatment of upper respiratory tract infections from November to April were included. A data collection form that included question items on type of antibiotic, duration of treatment, medical conditions, other medications, performance of culture tests, sensitivity results, number of antibiotics and duration, adverse drug reactions and use of generic versus trade names. The number of prescriptions was 300. SPSS was used for statistical analysis. Forty three percent who received antibiotics were less than 18 years of age, 20% were between 18–30 years and 16% were above 30 years. Males were 43% and females 47%. Most patients had one course of antibiotics (94%), 6% had two courses. There was no documentation of adverse drug reactions. In 45% of prescriptions generic names were used and 55% used trade names. The duration of treatment for 66% of patients was five days, 7 days for (21%) and less than 5 days in (9%). No data on antibiotic sensitivity was available. Co-amoxiclav accounted for (62%), followed by amoxicillin 37% and azithromycin 12%. Antibiotics were largely prescribed to children. Antibiotics ranked 5th highest prescribed drugs in infants (Al Khaja et al., 2006). In our study most prescriptions were empirical. More co-amoxiclav prescribing compared with amoxicillin indicates resistance. The trend of prescribing beta lactam antibiotics for upper respiratory tract infections by primary care physicians has also been observed in other parts of the Arabian Gulf. (Al Khaja et al., 2008). References: Al Khaja KA et al. P J Trop Pediatr. 2006 Dec; 52 (6): 390–393. Al Khaja KA et al, Pharmacoepidemiol Drug Saf. 2008 Mar 5.