Selectivity of interaction of spin-labelled lipids with peripheral proteins bound to dimyristoylphosphatidylglycerol bilayers, as determined by ESR spectroscopy.

The selectivity of interaction between spin-labelled lipids and the peripheral proteins, apocytochrome c, cytochrome c, lysozyme and polylysine has been studied using ESR spectroscopy. Derivatives of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (CL) and diacylglycerol (DG) spin-labelled at the 5-C atom position of the sn-2 chain were used to study the association of these proteins with bilayers of dimyristoylphosphatidylglycero. Binding of the proteins increased the outer hyperfine splitting in the ESR spectra of the lipid spin labees to an extent which depended both on the spin-labelled lipid species involved and on the particular protein. The order of selectivity for apocytochrome c follows the sequence: PI−>CL−≈DG PS−>PC±>PG−>PE±. The selectivity pattern for cytochrome c is: PI−>PG−>CL−>DG PS−≈PC±>PE±; for lysozyme is: CL−>PG−>DG PE−>PC±PS−>PI−; and that for polylysine is: CL−>PS−⩾PG−>PI−>PC±>DG PE+-. The overall strength of interaction is in the order lysozyme>cytochrome c>apcoytochrome c, for equivalent binding, and the spread of the selectivity for the different proteins is in the reverse order. Assuming fast exchange for the ESR spectra of the 5-C atom labelled lipids, the relative association constants of the different labels with the different proteins have been estimated

Conformational and ion-binding properties of cyclolinopeptide A isolated from linseed

The conformation of the cyclic nonapeptide from linseed, cyclolinopeptide A in methanol and in acetonitrile has been elucidated by one- and two-dimensional nuclear magnetic resonance. The molecule is folded in a ß-turn conformation. Cyclolinopeptide A interacts and weakly complexes with Tb3+ (a Ca2+ mimic ion) with the metal ion positioned proximally to the Phe residue, but with no substantial structural alteration upon metal binding. Cyclolinopeptide A is also seen to aid the translocation of Pr3+ (another Ca2+ mimic) across unilamellar liposomes. However, cyclolinopeptide A does not phase transfer or act as an ionophore of calcium ion myself. Experiments using lanthanide ions thus do not necessarily indicate any ionophoretic ability of the complexone towards calcium ions

Location of valinomycin in lipid vesicles

The location of the cyclododecadepsipeptide, valinomycin in vesicles formed from two synthetic lipids is studied by differential scanning calorimetry, spin-label partitioning electron paramagnetic resonance and [1H]-nuclear magnetic resonance. The results show that valinomycin is located near the head group region of dipalmitoyl phosphatidyl choline vesicles and in the hydrophobic core of the dimyristoyl phosphatidyl choline vesicles in the liquid crystalline phase

Mechanisms of transmembrane cation transport studied by nuclear magnetic resonance spectroscopy

Several molecules like ionophores, vitamins, ion-binding cyclic peptides, acidic phospholipids, surfactants are known to expose the inner side of vesicles, to the externally added cations. Whereas ionophores and certain other systems bring about these changes by a selective transport (influx) of the cation by specialized mechanisms known as the carrier and channel mechanism, other systems cause lysis and vesicle fusion. These systems have been successfully studied using1H,31 P and13C nuclear magnetic resonance spectroscopy after the demonstration, fifteen years ago, of the ability of paramagnetic lanthanide ions to distinguish the inside of the vesicle from the outside. The results of these'nuclear magnetic resonance kinetics' experiments are reviewed

Effects of domain connection and disconnection on the yields of in-plane bimolecular reactions in membranes

It has recently been shown (Vaz, W.L.C., E.C.C. Melo, and T.E. ThomPson. 1989. Biophys. J. 56:869-875; 1990. Biophys. J. 58:273-275) that in lipid bilayer membranes in which ordered and disordered phases coexist, the ordered phase can form a two-dimensional reticular structure that subdivides the coexisting disordered phase into a disconnected domain structure. Here we consider theoretically the yields of bimolecular reactions between membrane-localized reactants, when both the reactants and products are confined to the disordered phase. It is shown that compartmentalization of reactants in disconnected domains can lead to significant reductions in reaction yields. The reduction in yield was calculated for classical bimolecular processes and for enzyme-catalyzed reactions. These ideas can be used to explain certain experimental observations.NIGMS NIH HHS [GM-23573, GM-14628]info:eu-repo/semantics/publishedVersio

Insect Antifeedants And Growth Inhibitors From Azadirachta Indica And Plumbago Zeylanica

Solvent extracts of dlfferent parts of the neem tree (Azadlrachta lndica A. 3uss) were bioassayed for thelr antlfeedant properties agalnst flrst- and thlrd-instar larvae of Mythimna reparata. Fraction 'C', obtalned from an ethanollc extract of the shade-drled neem seeds (Hyderabad, India) was the most effective phagodeterrent under glasshouse, laboratory and, to a 11mlted extent, fleld conditions. Fractlon 'C' was further purlfled to fractlon 'M', whlch was subjected to column chromatography (silica gel) result~ng In IG fractions, AI-l to AI-14. Fractions AI-9, AI-10 and AI-11 were found to be b~olog~calalyc tive, reduclng leaf feedlng and larval weight consrderably. None of the treated larvae whlch fed on leaf dlscs treated wlth the fractions and extracts 'C' and 'M' could pupate. The retentlon tlme of the malor component of AI-10 on a p BONDAPAK C18 10 u, 3.9 mm ID, 30 cm column (hlgh performance liquid chromatography) was close to that of azadlrachtln. Spectral lnvestlgatlons revealed that this major component of A1 1s dlfferent from the antifeedants reported earher from A, indica and Melia azedarach. It has been des~gnated as vepaol (the Telugu name for neem IS vepa), and some aspects of 11s structure are discussed. Studles relatlng to the effectiveness of fraction 'G' on the oriental rmyworm, Mythrmna reparata, and other sorghum pests are presented

Dicationic Alkylammonium Bromide Gemini Surfactants. Membrane Perturbation and Skin Irritation

Dicationic alkylammonium bromide gemini surfactants represent a class of amphiphiles potentially effective as skin permeation enhancers. However, only a limited number of studies has been dedicated to the evaluation of the respective cytotoxicity, and none directed to skin irritation endpoints. Supported on a cell viability study, the cytotoxicity of gemini surfactants of variable tail and spacer length was assessed. For this purpose, keratinocyte cells from human skin (NCTC 2544 cell line), frequently used as a model for skin irritation, were employed. The impact of the different gemini surfactants on the permeability and morphology of model vesicles was additionally investigated by measuring the leakage of calcein fluorescent dye and analyzing the NMR spectra of 31P, respectively. Detail on the interaction of gemini molecules with model membranes was also provided by a systematic differential scanning calorimetry (DSC) and molecular dynamics (MD) simulation. An irreversible impact on the viability of the NCTC 2544 cell line was observed for gemini concentrations higher than 25 mM, while no cytotoxicity was found for any of the surfactants in a concentration range up to 10 mM. A higher cytotoxicity was also found for gemini surfactants presenting longer spacer and shorter tails. The same trend was obtained in the calorimetric and permeability studies, with the gemini of longest spacer promoting the highest degree of membrane destabilization. Additional structural and dynamical characterization of the various systems, obtained by 31P NMR and MD, provide some insight on the relationship between the architecture of gemini surfactants and the respective perturbation mechanism

Charge Isomers of Myelin Basic Protein: Structure and Interactions with Membranes, Nucleotide Analogues, and Calmodulin

As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers

Effect of SGLT2 inhibitors on stroke and atrial fibrillation in diabetic kidney disease: Results from the CREDENCE trial and meta-analysis

BACKGROUND AND PURPOSE: Chronic kidney disease with reduced estimated glomerular filtration rate or elevated albuminuria increases risk for ischemic and hemorrhagic stroke. This study assessed the effects of sodium glucose cotransporter 2 inhibitors (SGLT2i) on stroke and atrial fibrillation/flutter (AF/AFL) from CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation) and a meta-Analysis of large cardiovascular outcome trials (CVOTs) of SGLT2i in type 2 diabetes mellitus. METHODS: CREDENCE randomized 4401 participants with type 2 diabetes mellitus and chronic kidney disease to canagliflozin or placebo. Post hoc, we estimated effects on fatal or nonfatal stroke, stroke subtypes, and intermediate markers of stroke risk including AF/AFL. Stroke and AF/AFL data from 3 other completed large CVOTs and CREDENCE were pooled using random-effects meta-Analysis. RESULTS: In CREDENCE, 142 participants experienced a stroke during follow-up (10.9/1000 patient-years with canagliflozin, 14.2/1000 patient-years with placebo; hazard ratio [HR], 0.77 [95% CI, 0.55-1.08]). Effects by stroke subtypes were: ischemic (HR, 0.88 [95% CI, 0.61-1.28]; n=111), hemorrhagic (HR, 0.50 [95% CI, 0.19-1.32]; n=18), and undetermined (HR, 0.54 [95% CI, 0.20-1.46]; n=17). There was no clear effect on AF/AFL (HR, 0.76 [95% CI, 0.53-1.10]; n=115). The overall effects in the 4 CVOTs combined were: Total stroke (HRpooled, 0.96 [95% CI, 0.82-1.12]), ischemic stroke (HRpooled, 1.01 [95% CI, 0.89-1.14]), hemorrhagic stroke (HRpooled, 0.50 [95% CI, 0.30-0.83]), undetermined stroke (HRpooled, 0.86 [95% CI, 0.49-1.51]), and AF/AFL (HRpooled, 0.81 [95% CI, 0.71-0.93]). There was evidence that SGLT2i effects on total stroke varied by baseline estimated glomerular filtration rate (P=0.01), with protection in the lowest estimated glomerular filtration rate (45 mL/min/1.73 m2]) subgroup (HRpooled, 0.50 [95% CI, 0.31-0.79]). CONCLUSIONS: Although we found no clear effect of SGLT2i on total stroke in CREDENCE or across trials combined, there was some evidence of benefit in preventing hemorrhagic stroke and AF/AFL, as well as total stroke for those with lowest estimated glomerular filtration rate. Future research should focus on confirming these data and exploring potential mechanisms