31P NMR analysis of the surface homogeneity of mixed sphingomyelin-phosphatidylcholine vesicles

The surface compositional symmetry of a mixed sphingomyelin-phosphatidylcholine vesicle has been studied by ³¹P NMR spectrometry. The molecules on the outer vesicle surface could be distinguished from the molecules on the inner vesicle surface by utilization of the shift reagent, Eu³⁺. This polyvalent cation interacts with the outer surface phosphate groups and, as a result, shifts these molecules upfield. Analysis of the data obtained indicates that a slight excess of sphingomyelin is present on the outer surface of the vesicle, compared to phosphatidylcholine, which appears to have a slight preference for the inner surface.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Trans-bilayer pseudocontact shifts produced by lanthanide ions in the 1H and 31P-nmr spectra of phospholipid vesicular membranes and their temperature variation

1. Shifts in the ¹H and ³¹P-nmr signals originating from the outer and inner phosphorylcholine head-groups and from the lipid acyl chains are observed when phosphatidylcholine vesicles are treated with increasing extravesicular concentrations of the lanthanides Eu³⁺, Pr³⁺, Yb³⁺, and Dy³⁺. 2. The addition of KNCS to increase the binding of the lanthanide ions to the outer head-groups is used to demonstrate that the intravesicular group shifts are not caused by bulk susceptibility effects. 3. The magnitude and direction of the observed shifts in the ¹H-nmr spectrum are shown to be consistent with (a) pseudocontact interaction of the paramagnetic lanthanide ions with the outer phospholipid head-groups, (b) current views of the conformation of the phosphatidylcholine head-group in the presence of lanthanides, and (c) a conservation of magnetic field within the vesicles due to their spherical nature. 4. Variation of the shifts with temperature are compared for egg phosphatidylcholine and dipalmitoyl phosphatidylcholine. The temperature variation in shifts is also used to study phase transitions in each monolayer and phase separations in mixed lipid systems.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Effect of bile salts on the absorption of glucose and oleic acid by the cestodes, Hymenolepis diminuta and H. microstoma.

The uptake of 2 mM 14C-glucose by H. diminuta during 1-min incubations was inhibited by addition of 10 mM sodium taurocholate (NaTC) to the incubation media. Preincubation in 10 mM NaTC for 30 min did not increase the inhibition, suggesting that the inhibition was competitive. This was confirmed with a standard Lineweaver-Burk experiment. Addition of 0.35 mM oleic acid to the NaTC micelles did not alter the level of inhibition. Sodium glycocholate (NaGC) did not inhibit the uptake of glucose by H. diminuta. The uptake of glucose by H. microstoma was also inhibited by NaTC, and was not affected by NaGC. H. diminuta absorbed 3.62 mumoles of oleic acid/g dry wt during 15-min incubations in mixed micelles of 10 mM NaTC and 0.35 mM oleic acid. The total uptake was determined as the sum of the ethanol extractable and nonextractable 3H-oleic acid. In 15 mM NaTC, the uptake of oleic acid was reduced by 50%; at 30 mM NaTC the uptake of oleic acid decreased by half again. Substituting NaGC for NaTC, the greatest uptake of oleic acid, 2.63 mumoles/g dry wt, was from mixed micelles of 15 mM NaGC and 0.35 mM oleic acid. Lesser amounts of oleic acid were absorbed from mixed micelles at 5 or 30 mM NaGC. H. microstoma exhibited a similar pattern of oleic acid uptake from mixed micelles with NaTC and NaGC. At all bile salt concentrations tested, H. microstoma absorbed more oleic acid than H. diminuta and incorporated more oleic acid into the nonextractable pool. The possible roles of bile salts in the absorption of oleic acid as indicated by the results herein are discussed.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.Key words: angiogenesis, endothelial progenitor cells, statin, SDF-1, migrationAngiogenesis is the process by which new vessels form in ischemic tissue. The cytokine Stromal Cell Derived Factor-1 (SDF-1) is released into the circulation in response to ischemia and is an initiating signal in the angiogenesis process. SDF-1 mobilizes bone marrow cells (BMC) by binding to the cell surface receptor CXCR4. BMCs then enter the circulation and migrate to the ischemic site following the SDF-1 gradient. On arrival, BMCs promote angiogenesis by providing cellular elements such as endothelial cells (EC) and perivascular cells and also by secreting signaling proteins that mature the angiogenesis process. BMC surface CXCR4 expression and the SDF-1/CXCR4 interaction are essential for BMC to home to the injured site.Cell-based strategies to improve neovascularization of ischemic tissue have been achieved by injecting mononuclear cells derived from either BM¹ or peripheral blood, directly into ischemic muscle,² or by mobilizing BM-MNC with cytokines³ or other drugs such as statins.^4–6Statins are 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors and are primarily used to lower circulating cholesterol levels. In addition to reducing cholesterol synthesis, inhibition of the mevalonate pathway prevents synthesis of isoprenoid intermediates including geranylgeranylpyrophosphate. Geranylgeranylation is important in the posttranslational modification of intracellular signaling proteins, including Rho GTPases. This mechanism underlies many of the pleiotropic effects including the ability of statins to stabilize endothelial nitric oxide synthase mRNA and increase nitric oxide biosynthesis. In fact, statins have been shown to protect against ischemic injury of the heart and stimulate angiogenesis in ischemic limbs of normocholesterolemic animals.^7,8 The mechanism of action of statins has been demonstrated via mobilization of BM endothelial progenitor cells (EPCs) and facilitation of EPC incorporation into the neovasculature through a phosphoinositide-3 (PI-3) kinase-dependent pathway.^4–6 Statins have also been reported to enhance EPC migration, augment EPC chemotaxis and inhibit EPC apoptosis both in vitro and in vivo.^4,9,10SDF-1, an 89-amino acid polypeptide, is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells.¹¹ SDF-1 was initially identified as a potent chemoattractant for lymphocytes and monocytes, and as an enhancer of B cell proliferation. SDF-1 is considered to be a key regulator of hematopoietic stem cell trafficking between BM and the peripheral circulation. SDF-1 is highly expressed in ischemic tissues.^12,13 Elevation of SDF-1 levels in peripheral blood results in BMC mobilization to the peripheral circulation with a concurrent decrease within the BM.¹⁴ SDF-1 not only mobilizes progenitor cells in BM but also directs them to the ischemic site by promoting cell migration and proliferation.^3,15 SDF-1 may generate a gradient similar to developmental morphogens during ischemia that provides the cues and directions for progenitor cell mobilization into peripheral blood and homing to ischemic tissues.^16,17 Furthermore, SDF-1 also reduces EPC apoptosis and enhances survival of the progenitor cells.^3,18 SDF-1, either delivered locally in its protein form,^3,19,20 or generated in situ via plasmid and viral vector-mediated gene expression,^10,21,22 enhances neovascularization by augmenting EPC recruitment into ischemic tissues.SDF-1 binding to its receptor CXCR4 on the cell surface provides essential signals for mobilization and homing of EPCs to the injured site.^23–25 SDF-1 binding with CXCR4 triggers internalization of CXCR4. This SDF-1/CXCR4 interaction results in elevation of cytoplasmic Ca²⁺ levels²⁶ and phosphorylation of PI-3 kinase and other protein kinases, e.g. Akt,²¹ MEK/ERK^27,28 and Janus kinase (JAK)-2.²⁹ Activation of Akt protein kinase further upregulates the activity of eNOS by increasing both eNOS expression and phosphorylation, which in turn catalyzes the production of nitric oxide (NO), an important signal molecule for vascular protection and remodeling.^21,26 Disruption of SDF-1/CXCR4 interaction impaired incorporation of EPC into sites of ischemia, and disturbed ischemic limb neo-vascularization.³⁰To explore if the combined use of a statin to mobilize BM EPCs and local overexpression of SDF-1 to augment EPC homing to ischemic muscle will result in superior angiogenesis versus use of either agent alone, we used the murine hindlimb ischemia model to determine the effects of Fluvastatin and SDF-1 on angiogenesis.¹⁰ Fluvastatin (5 mg/kg) was injected intra-peritoneally into the mice daily for 7 days to mobilize progenitor cells prior to ischemia-inducing surgery. NIH 3T3 cells transduced with the retroviral vector carrying SDF-1 gene were injected I.M. into the ischemic limb after surgery to locally deliver SDF-1 to ischemic muscle.²² The number of circulating EPCs increased 9–18 fold seven days post statin/SDF-1 treatment.Our data of single treatment with Fluvastatin are consistent with the previous reports that statins not only augment mobilization of progenitor cells by increasing circulating EPC originated from BM,^4,31 but also modulate their differentiation. We further give a new insight view of the mechanism for statin induced EPC mobilization. We found that statin induced activation of matrix metalloproteinases (MMP)-2 and -9 in EPC. The increased MMP activity could result in degradation of extracellular matrix.¹⁷ Progenitor cells will be such mobilized into circulation when the cellular attachment is reduced within the bone marrow niches. We show that statin alone can enhance the phosphorylation of Akt, promote EPC proliferation, migration and inhibit cell apoptosis in vitro. The proangiogenic effects of statin are also illustrated in vivo using a murine hind-limb ischemia model. In this model, Fluvastatin treatment results in more EPC in circulation, more BM derived progenitor cells in ischemic muscle, more cell proliferation, enhanced capillary formation, and diminished cell apoptosis; these effects end up in improved reperfusion versus control. The beneficial effects of statin on angiogenesis are independent of cholesterol since the total serum cholesterol level is not changed by Fluvastatin treatment under these experimental conditions.To be noted, the effect of statins on EPCs was found to be concentration dependent. EPC proliferation, migration and the inhibition of apoptosis are enhanced at low statin concentrations (10 nM and 100 nM) but are significantly inhibited at a higher statin concentration (1,000 nM). The toxic effect of statin at high concentration cannot be compensated by addition of SDF-1, indicating that Statin causes apoptosis in a pathway different from the pathway that SDF-1 uses to prevent EPC apoptosis. Increased apoptosis at the higher statin concentration could explain the reversed effect of stain in angiogenesis. These findings are consistent with the reports in which statins were found to have proangiogenic effects at low therapeutic concentrations but angiostatic effects at high concentrations, the latter effect being reversible by geranylgeranyl pyrophosphate.^32,33Combined statin and SDF-1 treatment significantly enhanced angiogenesis versus treatment with either reagent alone. More cell proliferation and less apoptosis were observed both in vitro and in vivo, along with increased cell migration and tube formation in vitro, and enhanced progenitor cell incorporation and higher capillary density in ischemic tissue in vivo. It is interesting to note that neither statin nor SDF-1 alone promotes EPC tube formation, but combined treatment results in significant EPC tube formation. These results suggest that SDF-1 and statin have different mechanisms of action with regards to the promotion of neovascularization. It is possible that each drug affects a specific subset of progenitor cells.The facilitative effect of both statin and SDF-1 on EPC proliferation and migration is involved with Akt phosphorylation and endothelial nitric oxide synthase (eNOS) activation. The mechanism by which statins promote angiogenesis is through, at least partly, improved nitric oxide bioavailability. Statins have been reported to induce eNOS mRNA stability³⁴ and eNOS activity through a PI3k/Akt dependent pathway.^31,35–37 However, neither eNOS mRNA/protein expression nor EPCs are reported to be essential for the therapeutic effect of Fluvastatin on hypoxia-induced pulmonary hypertension; Fluvastatin improved eNOS phosphorylation by a mechanism independent of Akt activation.³⁸ Our data favor a mechanism involving Akt phosphorylation since phosphorylated Akt is increased when EPCs are cultured in the presence of statin, and statin-enhanced EPC proliferation and migration were inhibited by the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.The angiogenic effects of SDF-1 also involve increased production of NO²⁶ as NO is essential for EC migration and angiogenesis. SDF-1α gene transfer has been shown to enhance eNOS activity.²¹ Our in vitro data confirmed the involvement of Akt and eNOS in SDF-1 mediated cell migration.¹⁰ Phosphorylated Akt is increased when EPCs are cultured in the presence of SDF-1. The facilitative effect of SDF-1 on EPC migration is blocked by both the Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the eNOS inhibitor L-NMMA. In contrast, L-NMMA does not reverse the inhibitory effect of SDF-1 on apoptosis, indicating that the inhibitory effect of SDF-1 on apoptosis is not mediated through NO.²²We also show that the expression of MMP-2 and MMP-9 was increased when EPCs were cultured in the presence of statin or SDF-1. MMPs are a family of proteolytic enzymes that degrade components of the extracellular matrix (ECM). Degradation of ECM is an essential step for cell mobilization and migration. Our data indicate that the novel effect of statin and SDF-1 on migration is through enhancement of MMP-2 and MMP-9 activity, resulting in ECM degradation, thus promoting progenitor cell mobilization and migration. Both Akt phosphorylation and expression of MMP-2 and MMP-9 in EPCs are further enhanced by combined treatment with statin and SDF-1. This result indicates that treatment of EPCs with either statin or SDF-1 as monotherapy results in a sub-maximal angiogenic response. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an optimal effect on progenitor cells (Fig. 1).Open in a separate windowFigure 1Effect of statins and SDF-1 on promoting angiogenesis. Statin enhances the phosphorylation of Akt with a yet undefined mechanism. SDF-1 binding with the G-protein coupled membrane receptor CXCR4 results in phosphorylation of protein kinases like PI3 kinase and Akt. Activation of Akt then upregulates the activities of MMPs and eNOS. NOS catalyze the synthesis of NO which is essential for the EPC migration. MMPs degrade extracellular matrix to initiate cell migration. Activation of Akt also prevents cell apoptosis. These reactions promote cell migration and proliferation and enhance EPC survival. EPCs from bone marrow are thus mobilized into circulation. The circulating EPC are homed into ischemia area in lure of SDF-1. EPCs contribute to neovascularisation, either directly by incorporation into endothelium and differentiation into endothelial cells or indirectly by differentiating into perivascular cells that provide physical support and secrete signaling proteins and structural enzymes enabling the angiogenesis process. The effects of statin partially overlap with that of SDF-1; and the combined use of two factors appears to have an additive/synergistic effect on progenitor cells.In summary, the combination of progenitor cell mobilization with statin and targeted recruitment into the ischemic bed by SDF-1 leads to improved blood flow in the ischemic limb versus treatment with either agent alone. Statin and SDF-1 therefore display synergism in promoting neovascularization. This result suggests that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia. However, the use of statins as a clinical modifier of angiogenesis is still unproven. A great number of patients have been treated with these drugs and if they were potently proangiogenic, one might expect to see an increased risk of tumors. However, there is no evidence that these drugs encourage tumor development. Likewise, there is no definitive evidence for an antiangiogenic, tumor-modulating action of statins. We await further studies with interest.     

Interaction of lysophosphatidylcholine with phosphatidylcholine bilayers. A photo-physical and NMR study

Several photo-physical methods together with ³¹P-NMR have been used to investigate the effect of lysophosphatidylcholine on phosphatidylcholine bilayers. ³¹P-NMR shows that the permeability of the vesicle to Eu³⁺ increases sharply above approx. 40% lysophosphatidylcholine: fluorescence-quenching studies also show this type of behavior. Similar sharp changes in vesicle properties are observed via the photo-physical technique at this lysophosphatidylcholine/phosphatidylcholine composition. Fluorescence spectra of pyrene and pyrene carboxaldehyde show that increasing lysophosphatidylcholine composition increases the polarity of the environments of these probes up to 40% lysocompound. Above this composition the photo-physical properties of the probes slowly revert to those characteristic of the micellar lyso-compound. The pyrene fluorescence lifetime, the fine structure of the fluorescence, and the case of formation of pyrene excimer in these bilayer mixtures suggest that pyrene complexes weakly with the charged nitrogen of the choline group of the phosphatidylcholine and that the physical state of the system has a striking effect on this complexation process. Similar experiments with simple quaternary compounds lend strong support to this suggestion. The studies monitor in several ways the effect of bilayer composition on movement of molecules in these systems. The degree or site of solubilization of carcinogens is also uniquely affected by composition.     

Interactions of inorganic mercury with phospholipid micelles and model membranes. A31P-NMR study

The binding of inorganic mercury Hg(II) to phospholipid headgroups has been investigated by phosphorus-31 nuclear magnetic resonance of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in water micellar and multilamellar phases. HgCl₂ triggers the aggregation of phospholipid micelles, leading to a lipid-mercury precipitate that is no longer detectable by high-resolution³¹P-NMR. The remaining signal area corresponds to micelles in the soluble fraction and is a non-linear function of the initial mercury-to-lipid molar ratio. Kinetics of micelle aggregation are exponential for the first 15 min and show a plateau tendency after 120 min. Apparent Hg(H) affinities for phospholipid headgroups are in the order: PE > PS > PC. The same binding specificity is observed when HgCl₂ is added to (1:1) mixtures of different micelles (PE + PC; PS + PC). However, mercury binding to mixed micelles prepared with two lipids (PE/PC or PS/PC) induces the aggregation of both lipids. Hg(II) also leads to a³¹P-NMR chemical shift anisotropy decrease of PC, PS and mixed (1:1) PE/PC multilamellar vesicles and markedly broadens PS spectra. This indicates that HgCl₂ binding forces phospholipid headgroups to reorient and that the concomitant network formation leads to a slowing down of PS membrane collective motions. Formation of a gel-like lamellar phase characterized by a broad NMR linewidth is also observed upon HgCl₂ binding to PE samples both in fluid (L) or hexagonal (H_II) phases. The PE hexagonal phase is no longer detected in the presence of HgCl₂. Mixed PE/PC dispersions remain in the fluid phase upon mercury addition, indicating that no phase separation occurs. Addition of excess NaCl leads to the appearance of the non-reactive species HgCl _inf4 ^sup2– and induces the reversal of all the above effects.Abbreviations A(t) time-dependence of peak area - A₄₀ peak area at t=40 min - 1/ rate of peak area decrease - isotropic chemical shift - isotropic chemical shift change - chemical shift anisotropy - DPPC dipalmitoylphosphatidylcholine - Hg(II) inorganic mercury - NMR nuclear magnetic resonance - pCl –log [Cl^–] - PC phosphatidylcholine - PE phosphatidylethanolamine - PL phospholipid - PS phosphatidylserine - R_i mercury-to-lipid molar ratio - MLV multilamellar vesicles - SUV small unilamellar vesicles     

Genes co-expressed with CPSAR1 identified using ATTED-II

Identification of interacting proteins will help to investigate further the relationship between CPSAR1 and the vesicle transport system or the ribosomes. Thus, we adopted a bioinformatic approach, using the publicly available Arabidopsis thaliana trans-factor and cis-element prediction database, ATTED-II (http://atted.jp/), to identify putative protein interactors. The proteins directly linked to CPSAR1 were almost exclusively nucleus encoded and several were involved in protein synthesis of which three were thylakoid localized. The list of putative interacting proteins does not exclude any of the previous proposed actions of CPSAR1 but encourage more detailed examination of the role of CPSAR1.Key words: Arabidopsis, chloroplast, co-expression, protein cargo, vesicle transportChloroplast protein targeting has been intensively studied.^1–4 Transport of non-proteinaceous material, such as lipids, to the thylakoid has not been studied to the same extent, although several reports do exist.^5–8 Since thylakoids do not produce lipids themselves any lipids present must have been transported from the production site, i.e., the envelope.^9,10 Experimental data support the theory of a vesicular transport system and it has been predicted that several proteins resembling those important in the cytosolic vesicle transport system are chloroplast localized.¹¹ Recently we confirmed that one of these proteins, CPSAR1, is located in the chloroplast and has features similar to the cytosolic COPII-related Sar1 protein, i.e., it is found at the donor membrane and in the vesicle, but not at the acceptor membrane. In addition, other research groups^12,13 have also found that CPSAR1 is chloroplast localized but refer to it as atOBGL or atObgM, respectively, since CPSAR1 shares high sequence similarity with proteins belonging to the Obg-family. The roles of Obg proteins are very diverse, but a role closely connected to ribosomes has been suggested for CPSAR1.¹² CPSAR1 being part of a vesicle system does not exclude it from having a role in relation to ribosomes. The existence of a coiled-coil motif and a GTP binding domain, in combination with the suggested role as part of a vesicle transport system, makes it a highly attractive idea that CPSAR1 interacts with other proteins.     

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and ³¹P, ¹³C, and ¹H nuclear magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5–, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and ¹³C- and ¹H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% -helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of -helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. Correspondence to: G. L. Mendz     

Phosphophoryn,an “acidic” biomineralization regulatory protein: Conformational folding in the presence of Cd(II)

The divalent cation-induced protein folding properties of the template macromolecule, bovine dentine phosphophoryn (BDPP), have been examined by ¹H/³¹P/¹³C/¹¹³Cd-nmr spectroscopy. Cd(II) was employed, exploiting the sensitivity of ¹¹³Cd-nmr to ligand-binding interactions and kinetics. Cation binding was studied over the stoichiometric range of 0–50 : 1 Cd(II) : protein (mole ratio), well below the range of Cd(II) concentration required to induce protein precipitation. The stepwise titration of divalent cation-depleted phosphophoryn at pH 7.2 in H₂O/D₂O with ¹¹³CdCl₂ revealed that (PSer)_n, (PSerAsp)_n, and (Asp)_n polyelectrolyte cation-binding domains undergo two major transitions in their secondary and tertiary structures: the first transition, occurring between 1 : 10 and 1 : 1 Cd(II) : protein stoichiometry, and the second, between 10 : 1 and 50 : 1. By monitoring the amide NH intensities, ³¹P-nmr chemical shift, and ¹³C Asp-C, resonances, it was concluded that Cd(II) ions exhibit a binding-site preference for polyelectrolyte cation-binding domains, in the order This preference correlates with the degree of negative charge density for each sequence motif. Accompanying the backbone conformational transitions at the polyelectrolyte regions were conformational transitions in the flanking hinge domains, indicating that the hinge domains participate in the folding of the phosphophoryn molecule as divalent cation binding occurs at the polyelectrolyte domains. We were unsuccessful in detecting phosphophoryn-bound Cd (11) species by ¹¹³Cd-nmr because of chemical exchange modulation. However, using a smaller 21-residue peptide mimetic of phosphophoryn, we have observed three stoichiometric-dependent ¹¹³Cd resonances that differ in terms of the oxoanion coordination number. Our observation of multiple Cd(II) species in the presence of the peptide supports our contention that Cd(II) has many chemically distinct coordination sites on phosphophoryn, each in multiple equilibria with H₂O, Cl^?, and side-chain oxoatoms. © 1994 John Wiley & Sons, Inc.     

Analysis of phospholipase C (Bacillus cereus) action toward mixed micelles of phospholipid and surfactant.

The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K_s^A. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, K_m^B. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 10³ μmol min^?1 (mg of protein)^?1. K_s^A is about 2 mm and K_m^B is 1 to 2 × 10^?10 mol cm^?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A₂ and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.     

Optically stimulated luminescence in LiCaAlF6:Eu2+ phosphor

Results on optically stimulated luminescence (OSL) in LiCaAlF₆:Eu²⁺ are reported. Continuous wave OSL signal as recorded using blue (470 nm) stimulation was found to be ~31% that of standard phosphor lithium magnesium phosphate. The rate of OSL depletion for standard phosphor lithium magnesium phosphate is only three times less as compared with that of LiCaAlF₆:Eu²⁺. Strong photoluminescence (PL) in the near ultraviolet region is observed for LiCaAlF₆:Eu²⁺ with the characteristic Eu²⁺ emission at 369 nm for 254 nm excitation. The thermoluminescence (TL) glow peak for LiCaAlF₆:Eu²⁺ was observed at around 180°C. The glow peak was about six times more intense compared with the dosimetric peak of the well known thermoluminescence dosimetric (TLD) phosphor LiF‐TLD 100. Thus this phosphor deserves much more attention than it has received until now and may be useful as a dosimetric material in radiation dosimetry. Copyright © 2015 John Wiley & Sons, Ltd.     

Phosphophoryn,a biomineralization template protein: pH-dependent protein folding experiments

The protein folding behavior of a polyelectrolyte protein, bovine dentine phosphophoryn (BDPP), in the pH range of 1.82–11.0 has been investigated. One- and two-dimensional nmr spectroscopy has been utilized to obtain proton spin assignments for amino acid residues in D₂O and in H₂O. One-dimensional ³¹P-nmr experiments verify the existence of three separate classes of O-phosphoserine (PSer) resonances in BDPP (α, β, χ), representing three distinct PSer residue populations at pH 6.94. By means of pH titration and ¹H-nmr, five populations of Asp residues can be identified. Three of these populations exhibit secondary inflection points on their pH titration curves that correspond to an observed pK_a of 6.17–6.95. The presence or absence of secondary inflection points for Asp populations and the ³¹P-nmr chemical shift dispersion for the three PSer residue populations indicate that BDPP may be comprised of homologous (Asp-Asp)_n. (PSer-PSer)_n, and heterologous (PSer-Asp)_n sequences arranged into polyelectrolyte cluster regions. The pH titration also revealed that certain populations of Ser, Gly, and Pro residues in BDPP exhibit pH-dependent resonance frequency shifts. The “apparent” pK, for the transition points of these frequency shifts corresponds to either the pK of Pser monophosphatc ester and/or the pK_a of Asp COOH group of BDPP polyelectrolyte regions. On the basis of these transition points, we can assign four types of Ser, Gly, or Pro-containing “intervening” regions in BDPP, based on their sensitivity to protonation and deprotonation events occurring at (Asp)_n, (PSer)_n, or (PSer-Asp)_n anionic cluster regions that flank the intervening regions. Our ¹H-ninr experiments also reveal that BDPP assumes a folded conformation at low pH. As the pH increases, this conformation undergoes several unfolding transitions as the BDPP molecule assumes more open conformations in response to increased electrostatic repulsion between polyelectrolyte anionic regions in the protein. These folding-unfolding transitions are mediated by the intervening regions, which act as “hinges” to allow the polyelectrolyte regions to fold relative to one another. © 1994 John Wiley & Sons, Inc.     

Cellular Senescence Induced by Prolonged Subculture Adversely Affects Glutamate Uptake in C6 Lineage

Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60–180 min of l-[³H]glu incubation, LPC6 presented an intracellular [³H] 55 % lower than EPC6. Both cultures showed a time-dependent increase of intracellular [³H] reaching maximal levels at 120 min. Cultures incubated with d-[³H]asp showed a time-dependent increase of [³H] until 180 min. Moreover, LPC6 have a d-[³H]asp-derived intracellular [³H] 30–45 % lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50 % of l-[³H]glu-derived intracellular [³H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high β-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.     

Kinetics of Vibrio cholerae sialidase action on gangliosidic substrates at different supramolecular-organizational levels

G_d1a, G_d1b and G_t1b gangliosides were dispersed in the following membrane-mimicking systems: (a) homogeneous micelles; (b) mixed micelles with G_m1 ganglioside (which is resistant to the enzyme action), Triton X-100 or bovine serum albumin; (c) small unilamellar vesicles of egg phosphatidylcholine. The effect of dispersion on sialic acid release by Vibrio cholerae sialidase was studied. As reference substrates freely interacting with the enzyme the lipid-free carbohydrates of G_d1a and 3′-sialosyl-lactose were employed. The apparent V_max. of the enzyme was, with all the gangliosides, dependent on the type of ganglioside dispersion. It was lowest for homogeneous micelles and mixed micelles with ganglioside G_m1, and increased about 6-fold for ganglioside/bovine serum albumin lipoprotein micelles, 15-fold for mixed-ganglioside/Triton X-100 micelles (optimal molar ratio 1:7.5) and 30-fold for phosphatidylcholine vesicles containing 2.5 mol% ganglioside (this proportion was optimal for enzyme activity on the vesicles). For ganglioside G_d1a, the activity on Triton X-100 mixed micelles and on mixed vesicles was even greater (3- and 6-fold respectively) than that displayed on G_d1a lipid-free carbohydrate. With each of the used gangliosides the apparent K_m values were very similar values for homogeneous micelles and vesicular dispersions, but showed marked increases for Triton X-100 mixed micelles, approaching the values exhibited by reference oligosaccharides. Triton X-100 micelles and phosphatidylcholine vesicles did not appreciably alter the kinetics of sialidase action on 3′-sialosyl-lactose and on G_d1a lipid-free carbohydrate, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.     

Effect of lyophilization on liposomal encapsulation of salmon calcitonin

Purpose: The intent of this work was to assess the impact of lyophilization on the encapsulation of salmon calcitonin (sCT) into liposomes.

Methods: Four different liposomal formulations were investigated, i.e. DPPC:Chol:DSPE-PEG₂₀₀₀ (75:20:5 and 65:30:5) and DPPC:Chol (80:20 and 66.7:33.3). Lipid films were prepared and hydrated with loading buffer containing sCT and different concentrations of the cryoprotectant, trehalose dihydrate. The liposomes were lyophilized, reconstituted and extruded to obtain small unilamellar vesicles. Non-encapsulated sCT was separated by gel filtration. Non-lyophilized formulations and liposomes lyophilized without the cryoprotectant were used as controls. Liposomes were analyzed for particle size, polydispersity index, zeta-potential and encapsulation efficiency. ³¹P-NMR (phosphorous nuclear magnetic resonance spectroscopy) was performed on selected formulations.

Results: Post-lyophilization, no significant change in particle sizes and zeta-potentials were noted, regardless of the presence or absence of the cryoprotectant. Encapsulation efficiencies, however, increased following lyophilization, in both PEGylated (lyophilization control batch) and non-PEGylated liposomes (cryoprotectant batches only). ³¹P-NMR revealed the presence of two distinct vesicle populations – liposomes and micelles – in PEGylated formulation. The presence of micelles might be responsible for the observed encapsulation enhancement of sCT in the PEGylated formulation.

Conclusions: Lyophilization resulted in an increase in encapsulation efficiency of sCT in PEGylated liposomes, even in the absence of a cryoprotectant, due to presence of micellar vesicles.