Interaction of fluorescent probes with membranes of sarcoplasmic reticulum in AMP deamination

Gentamicin and ampicillin were dissolved in an L-amino acid solution especially prepared for newborn infants and infused intravenously over 24 h in 7 babies with serious neonatal surgical problems. Serum concentrations of the antibiotics were maintained rather constant and well above the minimal inhibitory concentration for most bacterial strains. One very sick newborn infant died with overwhelming Klebsiella pneumoniae septicemia. No signs of renal toxicity or ototoxicity were found. The serum amino acids remained within the normal range, except in 1 child with cytomegalovirus infection and liver insufficiency.     

113Cd-, 31P-NMR and fluorescence polarization studies of cadmium(II) interactions with phospholipids in model membranes

Cadmium(II) interactions with multilamellar vesicles of dimyristoyl (DM)- and dipalmitoyl (DP)-phosphatidylcholine (PC), -phosphatidylserine (PS), -phosphatidic acid (PA), -phosphatidylglycerol (PG) and -phosphatidylethanolamine (PE) have been investigated both from the metal and the membrane viewpoints, respectively, by solution 113Cd-NMR and diphenylhexatriene fluorescence polarization coupled with solid-state 31P-NMR. Results can be summarized as follows. (1) Strong cadmium binding to membrane phospholipids results in a decrease of the free Cd(II) 113Cd-NMR isotropic signal and because of slow exchange, in the NMR time scale, between free and bound cadmium pools, the lipid/water partition coefficients, Klw, of the Cd(II) species can be determined in the lamellar gel (fluid) phase. It is found Klw(DMPC) approximately Klw(EggPE) approximately 2+/-2 (2+/-2); Klw(DMPA)=392+/-20 (505+/-25); Klw(DMPG)=428+/-21 (352+/-17); Klw(DMPS)=544+/-27 (672+/-34). Cadmium interactions with membrane phospholipids are therefore electrostatic in nature and the phosphate moiety is proposed as a potential binding site. (2) The presence of Cd(II) stabilizes the gel phases of PG, PA and PS lipids and leads to suppression of the main phase transition for PA and PS. These effects are reduced upon increasing salinity to 0.5 M Cl- and abolished at 1.8 M Cl-, Cd(II) being removed from the membranes due to formation of soluble CdCln species. Moving the pH from 7 to 6 also decreases Cd(II) binding to PA, because of surface charge reduction. (3) Cadmium promotes the formation of isotropic 31P-NMR lines with PG systems and of a hexagonal phase on egg PE bilayers at 24 degreesC, suggesting dramatic membrane reorganization. Properties of cadmium and calcium interacting with phospholipid model membranes are compared, and the potential roles of these interactions in the molecular mechanisms of cadmium uptake and toxicity in cells are discussed.     

Chromosome location of genes encoding human blood groups

In this study the transactivation potential and DNA binding activities of p53 protein were examined following exposure of A2780 cells, a human ovarian carcinoma cell line, to the DNA damaging agent, cis-diamminedichloroplatinum II (cisplatin). The endogenous murine double minute-2 gene (mdm-2) was used to monitor the ability of p53 to transactivate genes. Northern analysis showed an induction of mdm-2 mRNA upon cisplatin treatment. It was further demonstrated, using an RNase protection assay, that the p53-responsive, mdm-2 promoter (P2) was activated in cisplatin-treated A2780 cells. However, when p53 protein DNA binding activity was analyzed, there was no detectable increase in p53 sequence-specific DNA binding activity during the period of time following DNA damage when mdm-2 mRNA was induced. Instead the increase in p53 protein observed in nuclear, cytoplasmic, and whole cell extracts correlated with a latent conformation of p53 that lacked sequence-specific DNA binding activity. At low doses of cisplatin, these latent pools of p53 increased in parallel with mdm-2 gene activation and were detectable as early as 4 h following cisplatin treatment. In vitro attempts to convert the latent p53 into an active, sequence-specific DNA binding conformation were unsuccessful. Even though cisplatin-induced p53 lacked sequence-specific DNA binding activity, it does possess an increased affinity for cisplatin-damaged duplex DNA molecules. This represents the first identification where cisplatin treatment induces a p53 protein, lacking sequence-specific DNA binding but with an increased affinity for platinated DNA molecules.     

Promotion of microtubule assembly by oligocations: cooperativity between charged groups

The rate and, to a lesser degree, the extent of microtubule assembly from rat brain tubulin is enhanced by oligocations such as polyamines, melittin, polybasic drugs, oligolysines, and oligoarginines. The effect is cooperative for degrees of polymerization up to seven for oligolysines and up to five for oligoarginines and is interpreted as an interaction with up to seven closely spaced anionic charges. Microtubules so formed appear to be normal by electron microscopy, and by salt, colchicine, and cold sensitivities. Lysyl residues in excess of seven (or five for arginine) in larger oligomers interact nearly noncooperatively. Separation of lysyl charges by intercalation of alanyl residues reduced assembly promoting potency for hexalysines. The cooperative portion of the response is most likely associated with the highly acidic extreme C termini of tubulin because their removal with limited subtilisin treatment markedly reduces oligolysine potency. However, some cooperative interactions with oligocations can also occur with more widely spaced anionic charges elsewhere in tubulin. The potential role of oligocations in the intracellular regulation of microtubule assembly is discussed.     

Anisotropy of photosynthetic membranes and the degree of fluorescence polarization

The degree of fluorescence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400-680 nm) and emission wavelengths (675-750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, PIN and PAN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization PIN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Qy (approximately 660-680 nm) absorption band of chlorophyll in vivo. With 670-680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts PIN is shown to be +5+/-1%. The excitation wavelength dependence studies of PIN indicate that the carotenoid and chlorophyll Qy transition moments tends to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).     

Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes

The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally, it is found that the binding capacity for the native protein is increased at low ionic strength to a value that is greater than that for complete surface coverage, and that corresponds more closely to neutralization of the effective charge (determined from the ionic strength dependence), rather than of the total net charge, on the protein. Electron spin resonance experiments with spin-labeled lipids indicate that this different mode of binding arises from a penetration or disturbance of the bilayer surface by the protein that may alleviate the effects of in-plane interactions under conditions of strong binding.     

A specific decrease of the fluorescence depolarization of perylene in muscle membranes from mice with muscular dystrophy

The microviscosity of erythrocyte membranes and muscle microsomes from age matched 6-week old control mice REJ 129 Dy/Dy, and mice with muscular dystrophy REJ 129 DY/DY has been estimated by measuring the fluorescence depolarization of perylene. There was no difference between the erythrocyte membranes. The muscle microsomes from dystrophic animals had about 20% lower values than the controls. The temperature dependence indicated that a transition occurs in both sets of muscle microsomes, but the transition temperature was lower in the dystrophic microsomes. Cholesterol, phospholipid and triglyceride analyses of the membranes showed no difference between the erythrocyte membranes. The largest difference in the muscle microsomes was a two-fold increase in cholesterol level found in the dystrophic microsomes. No simple correlation could be made between the lipid analysis and the microviscosity measurements. Since the change in microviscosity is found in membranes isolated from the tissue primarily affected by the dy gene, we suggest that the change in microviscosity may be important in the development of the disease.     

Interaction of the lantibiotic nisin with membranes revealed by fluorescence quenching of an introduced tryptophan

Nisin is a lantibiotic produced by strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of gram-positive bacteria. To aid understanding of its mode of action, the interaction of nisin with vesicles of differing phospholipid composition were investigated by fluorescence techniques, using a variant of nisin in which the isoleucine at position 30 was replaced by a tryptophan residue. Activity of the site-directed variant containing tryptophan was established to be similar to that of the wild-type peptide. Fluorescence experiments showed a blue shift of the emission wavelength maximum in the presence of lipid vesicles, indicating that the tryptophan residue enters a more hydrophobic environment. Quenching experiments with aqueous and membrane-restricted quenchers (iodide and spin-labelled lipids, respectively) both confirmed a non-aqueous environment for the Trp30 residue, and implied that the residue resides between 0.36 nm and 0.52 nm from the centre of the membrane, depending on the lipid identity. The results clearly demonstrate that nisin interacts strongly with the hydrophobic phase of lipid vesicles. This interaction is stronger in the presence of negatively charged lipids suggesting their importance in the functional interaction of nisin with membranes.