Simona Lobasso

The role of 3-dimethylaminopropylamine and amidoamine in contact allergy to cocamidopropylbetaine

Since it has been found that all subjects with contact allergy to cocamidopropylbetaine (CAPB) have positive reactions to 3‐dimethylaminopropylamine (DMAPA), and reports have appeared in literature of the sensitizing action of amidoamine in products containing CAPB, we aimed to verify the possibility that pure amidoamine may have a sensitizing role in subjects with positive reactions to CAPB. To this end, in 10 patients with contact allergy to a commercial CAPB, we tested DMAPA 1% aq. and a pure amidoamine in concentrations ranging from 0·5% aq. to 0·1% aq. The study showed that all patients with positive reactions to DMAPA reacted to amidoamine at 0·5% and 0·25% aq., while 4 of the 10 also had positive reactions to amidoamine at 0·1% aq. We consider that simultaneous allergic reaction to DMAPA and amidoamine represents cross‐reactivity and hypothesize that DMAPA is in fact the true sensitizing substance, while amidoamine, which may in any case release DMAPA in vivo as a result of enzymatic hydrolysis, may favour the transepidermal penetration of the sensitizing agent. In addition, we advise that testing of CAPB be suspended, because, as suggested by chemico‐structural analyses and demonstrated in vivo, when thoroughly purified, it no longer has a sensitizing action.

25 Characterization of Lipids of Halophilic Archaea

Publisher Summary One- or two-dimensional thin-layer chromatography (TLC) is a useful technique for the rapid characterization of the lipids present in halophilic archaeal isolates. Mass spectrometry and nuclear magnetic resonance (NMR) spectroscopic techniques are powerful analytical methods in studies of structural characterization of lipid mixtures, isolated lipid components, or chemical degradation products of these lipids. The study of lipid composition of archaeal extreme halophilic micro-organisms is of particular interest not only for its relevance for taxonomy, but also to shed light on membrane assembly and function and for the presence of a large variety of unusual structures of potential biotechnology applications. Lipids that are largely in hydrophobically associated form may be extracted with relatively non-polar solvents, such as ethyl ether chloroform or benzene. Preparative TLC of total lipid extracts of extreme halophilic Archaea can be carried out on silica gel plates, 20x20 cm and a 0.5-mm thick layer, while for analytical purposes TLC plates 10x20 cm and a 0.25-mm thick layer can be used.

Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi

The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the three-laminar structure of the cell wall, which differs significantly from that of other Haloarchaea.

LIGHT-DEPENDENT AND BIOCHEMICAL PROPERTIES OF TWO DIFFERENT BANDS OF BACTERIORHODOPSIN ISOLATED ON PHENYL-SEPHAROSE CL-4B

Abstract— We report a detailed description of the light‐dependent and biochemical properties of two different bands of isolated and nearly delipidated bacteriorhodopsin obtained from chromatography on phenyl‐Sepharose CL‐4B. The two bands (BR I and BR II) showed a number of markedly different spectroscopic and biochemical characteristics: different absorption maximums in the dark, different light/dark adaptations, different M decay kinetics, different stabilities, different responses to titration with alkali in the dark and different circular dichroism (CD) spectra. Organic phosphate contents of BR I and BR II were measured; we found that more than 90% of purple membrane organic phosphate was removed in the course of chromatography and that the phospholipid/protein molar ratio was always higher in BR I than in BR II. In many functional aspects (high stability, response to light adaptation, spectral changes in the dark by alkali addition and bilobate CD spectrum) the first band appeared to be similar to the purple membrane. We suggest that the functional differences between the two bands depend on the fact that the first band (BR I) contains mostly bacteriorhodopsin aggregates corresponding to purple membrane trimers, while the second band (BR II) contains only bacteriorhodopsin monomers.

MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine

A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extraction/separation steps, is described. The purple membrane isolated from the extremely halophilic archaeon Halobacterium salinarum was selected as model membrane. Lyophilized purple membrane were grinded with 9-aminoacridine (9-AA) as dry matrix, and the powder mixture was crushed in a mechanical die press to form a thin pellet. Small pieces of the pellet were then attached to the MALDI target and directly analyzed. In parallel, individual archaebacterial phospholipids and glycolipids, together with the total lipid extract of the purple membrane, were analyzed by MALDI-TOF/MS using 9-AA as the matrix in solution. Results show that 9-AA represents a suitable matrix for the conventional MALDI-TOF/MS analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized archaebacterial membranes. This method might be of general application, offering the advantage of quickly gaining information about lipid components without disrupting or altering the membrane matrix.

Lipidomic Analysis of Porcine Olfactory Epithelial Membranes and Cilia

The use of the matrix 9-aminoacridine has been recently introduced in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of both anionic and cationic phospholipids. In the present study, we take advantage of this technique to analyze the lipids of porcine olfactory mucosa and a membrane fraction enriched in cilia. Thin-layer chromatography (TLC) and 31P-NMR analyses of the lipid extracts were also performed in parallel. MALDI-TOF-MS allowed the identification of lipid classes in the total lipid extract and individual lipids present in the main TLC bands. The comparison between the composition of the two lipid extracts showed that: (1) cardiolipin, present in small amount in the whole olfactory mucosa lipid extract, was absent in the extract of membranes enriched in olfactory cilia, (2) phosphatidylethanolamine species were less abundant in ciliary than in whole epithelial membranes, (3) sulfoglycosphingolipids were detected in the lipid extract of ciliary membranes, but not in that of epithelial membranes. Our results indicate that the lipid pattern of ciliary membranes is different from that of whole-tissue membranes and suggest that olfactory receptors require a specific lipid environment for their functioning.

ROLE OF PALMITIC ACID ON THE ISOLATION AND PROPERTIES OF HALORHODOPSIN

Purified halorhodopsin was isolated from Halobacterium halobium as previously described (Duschl, A. et al. (1988) J. Biol. Chem. 263, 17016-17022). Two purple bands were eluted from phenyl-Sepharose column, indicating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography/mass spectrometry we could identify palmitate (which is only a minor lipid component of archaeal cells) among lipids associated with purple fractions. Typically the palmitate content of the first eluted band was higher than that of the second, indicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present depending on the fraction analysed. Very little or no palmitate was released from denatured halorhodopsin. By adding palmitate to buffers used in the phenyl-Sepharose chromatography, only one sharp purple band was collected, corresponding to the less retained halorhodopsin fraction. Pentadecanoic fatty acid could also affect the halorhodopsin chromatography. Chromatography of halorhodopsin in the presence of beta-mercaptoethanol showed only one band, corresponding to the more retrained halorhodopsin form. The two halorhodopsin fractions had different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of decay of the absorbance at 570 nm upon illumination. By following the decay of the absorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pKa values of deprotonation.

Force measurements on natural membrane nanovesicles reveal a composition-independent, high Young's modulus

Mechanical properties of nano-sized vesicles made up of natural membranes are crucial to the development of stable, biocompatible nanocontainers with enhanced functional, recognition and sensing capabilities. Here we measure and compare the mechanical properties of plasma and inner membrane nanovesicles ∼80 nm in diameter obtained from disrupted yeast Saccharomyces cerevisiae cells. We provide evidence of a highly deformable behaviour for these vesicles, able to support repeated wall-to-wall compressions without irreversible deformations, accompanied by a noticeably high Youngs modulus (∼300 MPa) compared to that obtained for reconstituted artificial liposomes of similar size and approaching that of some virus particles. Surprisingly enough, the results are approximately similar for plasma and inner membrane nanovesicles, in spite of their different lipid compositions, especially on what concerns the ergosterol content. These results point towards an important structural role of membrane proteins in the mechanical response of natural membrane vesicles and open the perspective to their potential use as robust nanocontainers for bioapplications.

Coupled TLC and MALDI-TOF/MS analyses of the lipid extract of the hyperthermophilic archaeon Pyrococcus furiosus.

The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

Archaebacterial lipid membranes as models to study the interaction of 10-N-nonyl acridine orange with phospholipids.

The dye 10-N-nonyl acridine orange (NAO) is used to label cardiolipin domains in mitochondria and bacteria. The present work represents the first study on the binding of NAO with archaebacterial lipid membranes. By combining absorption and fluorescence spectroscopy with fluorescence microscopy studies, we investigated the interaction of the dye with (a) authentic standards of archaebacterial cardiolipins, phospholipids and sulfoglycolipids; (b) isolated membranes; (c) living cells of a square-shaped extremely halophilic archaeon. Absorption and fluorescence spectroscopy data indicate that the interaction of NAO with archaebacterial cardiolipin analogues is similar to that occurring with diacidic phospholipids and sulfoglycolipids, suggesting as molecular determinants for NAO binding to archaebacterial lipids the presence of two acidic residues or a combination of acidic and carbohydrate residues. In agreement with absorption spectroscopy data, fluorescence data indicate that NAO fluorescence in archaeal membranes cannot be exclusively attributed to bisphosphatidylglycerol and, therefore, different from mitochondria and bacteria, the dye cannot be used as a cardiolipin specific probe in archaeal microorganisms.