Reevaluation of the phospholipid composition in membranes of adult human lenses by P NMR and MALDI MS

The phospholipid composition of adult human lens membranes differs dramatically from that of any other mammalian membrane. Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest lived cells in our body. This work reassesses previous assignments of phospholipid ³¹P NMR resonances in adult human lenses. The new assignments are based not only on chemical shifts but also on temperature coefficients. By addition of known phospholipids and examination by matrix-assisted laser desorption/ionization mass spectrometry, several misassigned resonances have been corrected. The revised composition reveals the possible presence of ceramide-1-phosphate and dihydroceramide-1-phosphate. Among glycerophospholipids, the most abundant one does not correspond to phosphatidylglycerol but may be due to the lysoform of alkyl-acyl analogs of phosphatidylethanolamine. Besides sphingophospholipids, adult human lens membranes contain significant amounts of ether (1-O-alkyl) glycerophospholipids and their corresponding lysoforms.     

Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition

The enhanced permeability of lipid bilayer membranes at their gel-to-liquid phase transition has been explained using a "bilayer lipid heterogeneity" model, postulating leaky interfacial regions between still solid and melting liquid phases. The addition of lysolipid to dipalmitoylphosphatidylcholine bilayers dramatically enhances the amount of, and speed at which, encapsulated markers or drugs are released at this, already leaky, phase transition through these interfacial regions. To characterize and attempt to determine the mechanism behind lysolipid-generated permeability enhancement, dithionite permeability and doxorubicin release were measured for lysolipid and non-lysolipid, containing membranes. Rapid release of contents from lysolipid-containing membranes appears to occur through lysolipid-stabilized pores rather than a simple enhancement due to increased drug solubility in the bilayer. A dramatic enhancement in the permeability rate constant begins about two degrees below the calorimetric peak of the thermal transition, and extends several degrees past it. The maximum permeability rate constant coincides exactly with this calorimetric peak. Although some lysolipid desorption from liquid state membranes cannot be dismissed, dialyzation above T(m) and mass spectrometry analysis indicate lysolipid must, and can, remain in the membrane for the permeability enhancement, presumably as lysolipid stabilized pores in the grain boundary regions of the partially melted solid phase.     

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.     

Critical micelle concentrations and stirring are rate limiting in the loss of lipid mass during membrane degradation by phospholipase A2.

In phospholipid membranes attacked by phospholipase A(2) (PLA(2)), accumulation of degradation products influences the binding affinity as well as the catalytic activity of PLA(2). Such accumulation in its turn depends on the rate of membrane degradation and the efflux of degradation products from the membrane, the latter being influenced by the stirring conditions in the system. This complicated process was investigated with a new ellipsometric technique for in situ measurement of membrane mass in a well-defined flow system. Planar phospholipid bilayers were formed on rotating silicon discs in buffer solution. After the addition of 0.05-100 ng/ml of PLA(2) (from Naja mocambique mocambique) to the buffer, mass desorption could be measured with a precision of 3-5 ng/cm(2), that is, about 1% of the surface mass of a single bilayer. Using radiolabeled phospholipids and thin-layer chromatography, it was verified that only the degradation products desorb from the membrane, which was confirmed by the desorption of mixtures of phospholipids, lysophospholipids, and fatty acids. The rotating disc allows the exact calculation of the mass transfer constant for transport-limited exchange of lipid between fluid and disc surface, as a function of rotation rate. By using the mass transfer constant, the critical micelle concentrations, and the mole fractions of products, desorption kinetics could be fully described. The amount of degraded phospholipid could be continuously monitored as the sum of the product mass still present in the membrane, as inferred from the desorption rate, and the mass already lost from the surface. It is concluded that ellipsometry is a suitable tool for studying the effects of PLA(2) on membranes.     

Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis

A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.     

羧甲基壳聚糖膜对皮肤成纤维细胞相容性研究

通过玻璃流延法制备不同分子质量的壳聚糖及羧甲基壳聚糖膜,以体外培养的人皮肤成纤维细胞作为对象,利用膜的浸渍液培养及膜表面直接培养法研究比较了两种多糖膜的细胞相容性.实验发现两种多糖膜的浸渍液对细胞均无毒性效应,生物安全性是小分子质量膜好于大分子质量膜,羧甲基壳聚糖膜好于壳聚糖膜.皮肤成纤维细胞在壳聚糖膜上的生长受到抑制,生长一段时间后细胞有聚集和脱落现象,而羧甲基壳聚糖膜上细胞能很好地贴附、生长,没有聚集和脱落现象.结果表明羧甲基壳聚糖膜具有比壳聚糖膜更优越的细胞相容性.     

Production of enzyme reactors after separation by non-denaturing two-dimensional electrophoresis and immobilization on membrane

Activities of carboxylesterase and malate dehydrogenase on membranes were retained after enzymes of mouse liver cytosol were separated by non-denaturing, two-dimensional electrophoresis (2-DE), stained using imidazole and zinc salts and electroblotted on to membranes. Furthermore, hydrolytic changes of phosphatidylcholine by the esterase were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after separation, and reversible staining and immobilization to membranes. Hydrolytic activity of the esterase on the membranes was 20% of the original activity of the tissue homogenate. The present method can be applied to the production of several types of enzyme reactors on membranes.     

Bacterial chemotaxis to naphthalene desorbing from a nonaqueous liquid

Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined. We studied the effect of chemotaxis by Pseudomonas putida G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL. Chemotaxis by wild-type P. putida G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains. While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source. The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation. The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants.     

Protein profiles of hatchery egg shell membrane

Background

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM).

Methods

Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins.

Results

Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic.

Conclusion

The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

     

Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes

Disaccharides such as sucrose and trehalose play an important role in stabilizing cellular structures during dehydration. In fact, most organisms that are able to survive desiccation accumulate high concentrations of sugars in their cells. The mechanisms involved in the stabilization of cellular membranes in the dry state have been investigated using model membranes, such as phosphatidylcholine liposomes. It has been proposed that the lyoprotection of liposomes depends on the depression of the gel to liquid-crystalline phase transition temperature (T(m)) of the dry membranes below ambient and on the prevention of membrane fusion by sugar glass formation, because both lead to leakage of soluble content from the liposomes. Since fusion is prevented at lower sugar/lipid mass ratios than leakage, it has been assumed that more sugar is needed to depress T(m) than to prevent fusion. Here, we show that this is not the case. In air-dried egg phosphatidylcholine liposomes, T(m) is depressed by >60 degrees C at sucrose/lipid mass ratios 10-fold lower than those needed to depress fusion to below 20%. In fact, T(m) is significantly reduced at mass ratios where no bulk sugar glass phase is detectable by Fourier transform infrared spectroscopy or differential scanning calorimetry. A detailed analysis of the interactions of sucrose with the P=O, C=O, and choline groups of the lipid and a comparison to published data on water binding to phospholipids suggests that T(m) is reduced by sucrose through a "water replacement" mechanism. However, the sucrose/lipid mass ratios necessary to prevent leakage exceed those necessary to prevent both phase transitions and membrane fusion. We hypothesize that kinetic phenomena during dehydration and rehydration may be responsible for this discrepancy.     

Matrix-assisted laser desorption mass spectrometry of rhodopsin and bacteriorhodopsin.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.     

Affinity elution of pyruvate kinase from phosphocellulose.

Pyruvate kinase from ascites tumour cells can be eluted from phosphocellulose by very low concentrations of phosphoenolpyruvate, fructose 1,6-bisphosphate, adenosine 5'-diphosphate and pyrophosphate, respectively. The appropriate limiting conditions for "facilitated desorption" of the enzyme from phosphocellulose by these ligands have been elaborated for achieving maximum selectivity and recovery in the process of its purification. This method has been designated as "affinity elution chromatography" owing to the specific interactions between a ligand as a constituent of the eluting medium with the adsorbed enzyme, which causes its selective desorption from the ion-exchanger. Affinity elution with phosphoenolpyruvate has been found to be very effective for preparation of the M-types of pyruvate kinase. A specific activity of 420 for an almost homogeneous preparation of pyruvate kinase from ascites tumour cells has maximally been obtained.     

Cardiolipin in hydrogenosomes: evidence of symbiotic origin

Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus, a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.     

Radiation inactivation of binding sites for high density lipoproteins in human fibroblast membranes

Radiation inactivation and target analysis were used to determine the molecular mass of the binding sites for high density lipoproteins (HDL) on membranes prepared from human fibroblasts. These membrane binding sites shared characteristics with the previously described HDL binding sites on whole fibroblasts in tissue culture. They exhibited the same affinity for HDL, the same ligand specificity, and the same sensitivity to proteolytic agents. They were also up-regulated by cholesterol loading of the cells. Kinetics of HDL dissociation from membrane binding sites could not be described by a single exponential function, indicating that HDL probably bind to multiple classes of sites on fibroblast membranes. After exposure to ionizing radiation, these sites decreased in number as an apparent single exponential function of radiation dose, corresponding to an average molecular mass of 16,000 +/- 1,000 Da, which is smaller than any known cell-surface receptor protein. These data indicate that HDL binding sites on fibroblast membranes are not "classical" receptors in that they are kinetically heterogeneous and small in molecular mass.     

Bacterial Chemotaxis to Naphthalene Desorbing from a Nonaqueous Liquid

Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined. We studied the effect of chemotaxis by Pseudomonas putida G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL. Chemotaxis by wild-type P. putida G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains. While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source. The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation. The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants.     

Massspectrometric analyses of transmembrane proteins in human erythrocyte membrane

It is difficult to understand the functional mechanisms of integral membrane proteins without having protein chemical information on these proteins. Although there have been many attempts to identify functionally important amino acids in membrane proteins, chemically and enzymatically cleaved peptides of integral membrane proteins have been difficult to handle because of their hydrophobic properties. In the present study, we have applied an analytical method to transmembrane proteins combining amino acid sequencing, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and liquid chromatography with electrospray ionization (LC/ESI) mass spectrometry. We could analyze most (97%) of the tryptic fragments of the transmembrane domains of band 3 as well as other minor membrane proteins. The peptide mapping of the transmembrane domain of band 3 was completed and the peptide mapping information allowed us to identify the fragments containing lysine residues susceptible to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and to 2,4-dinitrofluorobenzene (DNFB). This method should be applicable to membrane proteins not only in erythrocyte membranes but also in other membranes.     

Conformation of gramicidin a in water: inference from analysis of hydrogen/deuterium exchange behavior by matrix assisted laser desorption ionization mass spectrometry

Gramicidin A (the major component of gramicidin D) is a highly hydrophobic peptide with very little solubility in water. Hence, the conformation of this peptide has been extensively investigated in organic solvents and model membranes, but not in water. The peptide adopts a beta6.3-helical conformation in the monomeric and dimeric forms. We have investigated the conformation of gramicidin A in water by monitoring hydrogen-deuterium exchange by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Our results indicate that gramicidin A is monomeric and exists in a highly folded conformation. The metal ion bound forms are clearly discernible in the monomers. The presence of the dimeric form is not observed. It is unlikely this is due to the operating conditions or the method used, as both hetero- and homodimers in gramicidin D are detected when methanol is used as a solvent. The present study also establishes that the linear gramicidins retain a history of solvent environment when ions are generated by matrix-assisted laser desorption ionization and analyzed by time-of-flight.     

Are porphyrin mixtures favorable photodynamic anticancer drugs? A model study with combinatorial libraries of tetraphenylporphyrins

Reported here is the preparation of tetraphenylporphyrin libraries via efficient combinatorial solution-phase syntheses, their purification, and preliminary results from a bioorganic study on their uptake in liposome membranes. Libraries with up to 666 components were prepared with substituents including Br, CF3, Cl, CN, CO2Me, Et, F, OAc, and Ph. Further, a first example for the synthesis of more diverse libraries via a "latent libraries" approach is presented. This involves masking polar groups with lipophilic protecting groups. After purification of the latent library, the masking protecting groups are removed in a quantitative reaction that produces the library compounds as the only non-volatile components. Libraries were characterized by laser desorption time-of-flight mass spectrometry, NMR, and UV-vis spectroscopy. In vitro uptake into membranes of small sonicated liposomes was measured, both in terms of total porphyrin incorporation and in terms of structure-incorporation relationships. The latter were determined from isotopically-resolved laser-desorption mass spectra under conditions that yield quantitative results. Smaller libraries showed increased uptake of porphyrins bearing OH and CF3 substituents and lower uptake of ester-, alkyl-, and halide-bearing porphyrins. This structure-dependent selectivity disappears for larger libraries, however, where uniformly high uptake is observed, i.e., at a constant lipid:porphyrin ratio the total porphyrin incorporation is higher for libraries than for single compounds of similar polarity. We propose that the decreased concentration of individual compounds in large libraries is responsible for this effect. Membrane incorporation has previously been shown to correlate with photodynamic activity in vitro and in vivo.16 Therefore, these results may help to explain why photodynamic therapy of tumors, a modern anti-cancer treatment modality, is successfully performed with a complex mixture of porphyrins.     

Proteomic analysis of a lymphoma-derived cell line (DG75) following treatment with a demethylating drug: modification of membrane-associated proteins

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.