Absence Of Calcium Ions Research Articles

The selective adsorption mechanism of calcium ions/pullulan in the flotation separation of chalcopyrite from talc

The flotation separation of chalcopyrite from talc by use of pullulan and sodium butyl xanthate (SBX) under calcium ions was investigated in this study. The single mineral flotation shows that SBX collector had a good collecting ability for chalcopyrite while talc had a good natural floatability. The talc flotation was observably depressed while that of chalcopyrite was not affected in the presence of pullulan alone. With the addition of calcium ions. The depression of talc could be achieved with a small amount of pullulan. The artificial chalcopyrite/talc mixture flotation tests reveal that the addition of pullulan could realize the separation of chalcopyrite from talc in the absence and presence of calcium ions, and the dosage of pullulan with calcium ions was lower than that in the absence of calcium ions. Zeta potential measurements suggest pullulan was easily adsorbed onto the calcium ions treated talc surface, and the XPS tests further confirm that calcium ions could selectively adsorb onto the talc surface and increase its activated sites for hydrogen bonding with pullulan molecules. This study is expect to provide a novel polysaccharide depressant for the beneficiation of chalcopyrite from talc.

Rheological study of hybrid aqueous suspension of TEMPO-oxidized cellulose nanofibrils and graphene oxide

The incorporation of graphene oxide (GO) into a nanocellulose matrix has attracted considerable attention due to the unique advantages of both components. This study focuses on investigating the viscoelastic and flow properties of hybrid aqueous suspensions (2.00 w/v%), composed of TEMPO-oxidized cellulose nanofibrils (TOCNF) and GO at different TOCNF/GO weight ratios. To adjust the elastic properties of the hybrid suspensions, calcium ions are introduced, varying the concentration systematically to study their effects on the hybrid network structure. All blends exhibit shear-thinning behaviour and demonstrate elastic, gel-like properties. Notably, in the absence of calcium ions, the enhancement of elastic properties is more pronounced at higher GO fractions. Conversely, with the introduction of calcium ions, the enhancement of elastic properties becomes particularly important at higher TOCNF fractions. For the quantitative evaluation of these enhancements, we employ the logarithmic mixing rule. Significant positive deviations from the predictions of the logarithmic mixing rule are ascribed to the complex, concentration-dependent arrangement of cellulose nanofibrils and GO liquid crystals in the aqueous suspension, coupled with ionic crosslinking induced by calcium ions. The study aims to contribute to the understanding of the rheological behaviour of the TOCNF/GO hydrogel, showing potential advancements in various applications.Graphical abstract

Formation of Calprotectin Inhibits Amyloid Aggregation of S100A8 and S100A9 Proteins.

Calcium-binding S100A8 and S100A9 proteins play a significant role in various disorders due to their pro-inflammatory functions. Substantially, they are also relevant in neurodegenerative disorders via the delivery of signals for the immune response. However, at the same time, they can aggregate and accelerate the progression of diseases. Natively, S100A8 and S100A9 exist as homo- and heterodimers, but upon aggregation, they form amyloid-like oligomers, fibrils, or amorphous aggregates. In this study, we aimed to elucidate the aggregation propensities of S100A8, S100A9, and their heterodimer calprotectin by investigating aggregation kinetics, secondary structures, and morphologies of the aggregates. For the first time, we followed the in vitro aggregation of S100A8, which formed spherical aggregates, unlike the fibrillar structures of S100A9 under the same conditions. The aggregates were sensitive to amyloid-specific ThT and ThS dyes and had a secondary structure composed of β-sheets. Similarly to S100A9, S100A8 protein was stabilized by calcium ions, resulting in aggregation inhibition. Finally, the formation of S100A8 and S100A9 heterodimers stabilized the proteins in the absence of calcium ions and prevented their aggregation.

Diverse Fluorescence Resonance Energy Transfer Processes Originating from the Conformational Heterogeneity of the Calcium Indicator Yellow Cameleon YC3.60.

By combining time-resolved fluorescence spectroscopy and molecular biology, we have clearly elucidated the fluorescence mechanism of yellow cameleon YC3.60. YC3.60 is one of the most widely used fundamental calcium ion indicator proteins and is a tandem protein consisting of cyan fluorescent protein, calmodulin, and yellow fluorescent protein. Our results show that the conformational heterogeneity of YC3.60 leads to multiple FRET (fluorescence resonance energy transfer) processes occurring in a variety of structures. In the presence of calcium ions, FRET occurs in 75% of all YC3.60 molecules, and the intramolecular FRET faster than 20 ps is dominant among the three different FRET processes. Even in the absence of calcium ions, FRET occurs in 28% of YC3.60, where intra- and intermolecular FRET with a time constant of 160 ps is dominant among the four different FRET processes. YC3.60 with the immature chromophore, which is 25% of the total, would result in lower contrast on imaging. Conformational heterogeneity arises from the specific association of the two fluorescent proteins. It was clearly shown that the association of fluorescent proteins has a significant impact on the fluorescence mechanism. These observations lead to the promotion of research that elucidates the relationship between the higher-order structure and function of proteins.

Binding of calcium ions to putative antibiotics.

Calcium-dependent antibiotics represent a family of antibiotics with so far only one clinically approved member, daptomycin. Daptomycin binds to anionic bacterial membrane in a calcium-dependent fashion and induces lipid clustering. The synthesis and design of related substances is a promising source of antibiotics at a time where antibiotic resistance looms larger than ever. By developing a series of substances that bind to anionic membranes in a manner that depends on the presence of calcium ions, we hope to determine the minimum requirements for antibiotic activity. Calcium dependent antibiotics must bind calcium to function, and therefore their affinity for calcium is essential to understanding their efficacy. We determined the calcium affinity of novel compounds by pH titration, in conjunction with UV-vis spectroscopy, in the presence and absence of calcium ions. The apparent shift in the measured pKa can be used to determine the binding constant of calcium. We then compared the calcium binding constants thus obtained with those measured using a standard Ca2+-selective electrode. The next step will be to measure Ca2+-binding constants of lipophilic analogues when associated with a lipid bilayer. Conclusions regarding the suitability of a selection of compounds are discussed.

Cryo-EM structure of human Cx31.3/GJC3 connexin hemichannel.

Connexin family proteins assemble into hexameric channels called hemichannels/connexons, which function as transmembrane channels or dock together to form gap junction intercellular channels (GJIChs). We determined the cryo-electron microscopy structures of human connexin 31.3 (Cx31.3)/GJC3 hemichannels in the presence and absence of calcium ions and with a hearing-loss mutation R15G at 2.3-, 2.5-, and 2.6-Å resolutions, respectively. Compared with available structures of GJICh in open conformation, Cx31.3 hemichannel shows substantial structural changes of highly conserved regions in the connexin family, including opening of calcium ion-binding tunnels, reorganization of salt-bridge networks, exposure of lipid-binding sites, and collocation of amino-terminal helices at the cytoplasmic entrance. We also found that the hemichannel has a pore with a diameter of ~8 Å and selectively transports chloride ions. Our study provides structural insights into the permeant selectivity of Cx31.3 hemichannel.

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

Reversible platelet aggregation in the presence of calcium ions: mechanisms and potential value

Disorders in the functions of platelets – blood cells responsible for the blood clots formation and prevention – are observed as independent diseases, as a complication of cancer and hematological diseases or as a result of a therapy. Nowadays, a test of platelet aggregation by aggregometry is the only diagnostic method for assessing the platelets functions. There are several varieties of aggregometry, which differ both in the method of recording the formation of platelet aggregates and in the method of preparing platelets for the experiment. In most laboratories, it is customary to conduct aggregometry in platelet-rich plasma in the presence of citrate ions. In this case, the concentration of calcium ions in plasma decreases, it prevents the thrombin formation and the plasma coagulation. On the other hand, it has long been known that platelet aggregation in response to ADP in the presence of calcium ions (in blood plasma collected in heparin or hirudin tubes, also blocking plasma clotting) is reversible: after 1-5 minutes after the addition of the activator, the disaggregation begins until the light transmission of the solution (platelet concentration) returns to its original level. This phenomenon is called "reversible” platelet aggregation. Reversible aggregation (“disaggregation”) is sometimes observed in aggregometry of citrate plasma, especially in pediatric patients. However, it is usually not considered normal and is considered a sign of platelet dysfunction. This review considers the known mechanisms of disaggregation in the presence or absence of calcium ions in the medium. The role of secondary activation of platelets as a potential cause of irreversible aggregation is discussed, as well as possible versions for explaining the results of aggregometry, when reversible platelet aggregation is observed.

Use of a Fluorescent Antiscalant to Investigate Scaling of Reverse Osmosis Membranes

Knowledge of the scaling mechanism makes it possible to develop effective means to control scaling and improve the membrane performance, increasing the recovery. This paper presents new approaches to the study of the mechanism of scaling in the presence of polymeric inhibitors (antiscalants); the adsorption of antiscalant molecules on the crystal and membrane surfaces has been investigated. The relations of the antiscalant adsorption rates to the antiscalant dose and the calcium carbonate scaling rate have been revealed. For the first time, the inhibition process was “visualized” by using a fluorescent antiscalant containing a fluorescent moiety—a copolymer of N-allyl-4-methoxy-1,8-naphthalamide and acrylic acid (PAA-F1). The examination of the surfaces of crystals and membranes by scanning electron and fluorescence microscopy showed new unexpected results: the antiscalant is adsorbed on the membrane surface and on the surface of calcite crystals formed. Fluorescence turned out to be more intense and noticeable on the surface of crystal faces than inside the crystal. During the nucleation phase, the “dark” part of the crystal lattice is formed and then begins to be covered with a “luminous” layer of the fluorescent antiscalant, which blocks further crystal growth. During experiments with antiscalant solutions in distilled water, the antiscalant was found to be adsorbed on the membrane surface in the absence of calcium ions. In experiments in which the antiscalant was added into the original tap water, it was adsorbed on the surface of the resulting crystals, not on the membrane. The visualization of the crystal growth inhibition process opens up new possibilities for studying the mechanism of scaling and developing of new technologies to control scaling.

Investigation of antiscalant dosing influence on scaling process in reverse osmosis facilities and membrane surface adsorption

Introduction. Understanding of crystal growth mechanism enables to develop efficient tools to control scaling and improve the process of treatment using membranes and increasing the amount of filtrate output. This investigation is aimed at studying an antiscalant behaviour in reverse osmosis (RO) process when treating ground water. Experimental dependences of calcium carbonate scaling efficiency on antiscalant dosage were found. Rates of adsorption on crystal surface of scaling deposit and on membrane surfaces were compared. Dependences of rates of inhibitor adsorption on crystal surface versus scaling rates were determined. Inhibitor adsorption on RO membrane surfaces was studied. New approaches to studying crystal growth mechanism in the presence of polymeric inhibitors are presented. Materials and methods. In the course of experiments conducted with using inhibitor dissolved in distilled water, inhibitor sorption on membrane surface was observed in the absence of calcium ions. As to experiments with dosing the inhibitor in tap water, the inhibitor sorption on the membrane did not occur: the inhibitor was adsorbed on the surface of the scaling crystals. Results. Experimental relationships are obtained that show dependencies of calcium carbonate deposit growth rates versus RO facility filtrate output values in the presence of different antiscalants with their dose values of 3, 5 and 7 mg/l. The article shows that antiscalant dose value does not provide substantial influence on antiscalant efficiency when natural water with low hardness is treated in the RO facility. This permits substantial reduction of operational costs. It was also proved that inhibitor is not adsorbed on membrane surface during natural water treatment that also confirms efficiency of low antiscalant dosing. Conclusions. Low hardness values of natural water (3–5 mill equivalents per liter) demonstrate that antiscalant efficiencies do not depend on its dose. Rate of inhibitor adsorption on crystal surface during calcium carbonate deposition also increases with scaling rate increase. Rates of antiscalant consumption increase with antiscalant dose values. In natural water the dissolved antiscalant molecules are bonded with calcium ions therefore antiscalant does not react with membranes and is not adsorbed on membrane surface.

Determination of pH Effects on Phosphatidyl-Hydroxytyrosol and Phosphatidyl-Tyrosol Bilayer Behavior.

A robust method was developed to investigate the liposomal behavior of novel enzymatically-synthesized hydroxytyrosol and tyrosol phospholipids. Bilayer characteristic obtained by this method, including bilayer formation stability and adsorption properties, were explored using dynamic light scattering, zeta-potential measurements, and quartz crystal microbalance with dissipation monitoring (QCMD), respectively. Liposome diameters were found to typically increase from pH 5.5 to pH 10. Zeta potentials values, on the other hand, were found to be well below −25 mV at all pH conditions explored, with the lowest values (and thus, the best liposome stability) at pH 5.5 or pH 10. Quartz crystal microbalance with dissipation monitoring measurements demonstrated that 100% 1,2-dioloeoylphosphatidyl-hydroxytyrosol (DOPHT) liposomes adsorbed intact onto silica in buffer conditions at pH 5.5 and with no calcium, or at pH 7.5 with calcium (no adsorption was detected at pH 10). 1,2-Dioleoylphosphatidyl-tyrosol (DOPT) liposomes were shown to adsorb intact under buffer conditions only at pH 5.5 with and without calcium. 1,2-Dioleoylphosphatidyl-2-phenolethanol (DOPPE), in comparison, readily adsorbed intact at pH 7.5 without calcium and just slightly at pH 5.5 with calcium present, but formed a supported bilayer over hours at pH 5.5 in the absence of calcium ions.

Calmodulins from Schistosoma mansoni: Biochemical analysis and interaction with IQ-motifs from voltage-gated calcium channels

The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins – SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCav1B (residues 1923–1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels.

Adsorption of uranyl tricarbonate and calcium uranyl carbonate onto γ-alumina

The uranyl tricarbonate moiety, exemplified by UO2(CO3)34−, CaUO2(CO3)32−, and Ca2UO2(CO3)3(aq), is potentially expected to consist of mobile uranyl(VI) species in the aquifer of a deep geological repository for spent nuclear fuel. Time-resolved laser fluorescence spectroscopy (TRLFS) for the U(VI) surface speciation on γ-alumina was utilized to investigate the effect of various uranyl tricarbonate species on U(VI) adsorption. Under the prevailing carbonate conditions, the emission bands of the species of U(VI) adsorbed onto γ-alumina were found at 467.6 ± 0.1, 487.1 ± 0.1, 507.5 ± 0.1, 530.0 ± 0.1, and 552.8 ± 0.2 nm and were blueshifted compared with those of the U(VI)-hydroxo surface complexes. The shift to the shorter wavelengths was attributed to the spectral feature of bis- and/or tris-carbonato U(VI) complexes found in aqueous uranyl(VI) species and uranium(VI) minerals with two to three carbonate ions. The results obtained from attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy have confirmed the formation of U(VI)-carbonato surface complexation on γ-alumina in a bidentate manner. Based on identical spectroscopic properties, which were observed independently of the presence or absence of calcium ions in the uranyl tricarbonate system, a similar manner of U(VI) tricarbonate adsorption can be assumed, regardless of the absence or presence of calcium. According to the results from this work and comparison with previous studies, U(VI)-carbonato surface complexation tends to be favorable on mineral surfaces with a higher point of zero charge (pHPZC) such as alumina. Regarding the retention of U(VI) in the aquifer, the results presented in this work provide new insight and interpretation of U(VI) adsorption in carbonate-rich groundwater.

Lipopeptide daptomycin: Interactions with bacterial and phospholipid membranes, stability of membrane aggregates and micellation in solution

Daptomycin is a cyclic lipopeptide effective against multidrug Gram-positive bacteria. Despite having a net negative charge, it is selective against negatively charged bacterial membranes. It has been established that daptomycin's antibiotic activity is based on directly targeting the bacterial membranes and that this antibacterial activity depends on calcium ions. Importantly, however, both the precise role of ions and the physical mechanisms responsible for daptomycin's action remain poorly understood. We investigate these issues using three types of molecular dynamics simulations: umbrella sampling free energy calculations for a single daptomycin, unbiased simulations for daptomycin tetramers, and unbiased simulations of micellation of daptomycin both in the absence and presence of calcium ions. The simulations are in the excess of 4 μs. As the most important finding, we establish that binding of the calcium ions on the aspartic acid residues is the key to stabilizing daptomycin tetramers inside the model material membrane. These complexes are vital for daptomycin's antibacterial activity. In the absence of binding, the tetramer is not stable and moves slowly out of the membrane. We also demonstrate that in solution, micellation of daptomycin occurs both in the presence and absence of calcium ions, and discuss the similarities between the behaviors of daptomycin and amyloid peptides in membranes.

Roles of magnesium ions in vascular relaxation via calcium channel and cyclooxygenase pathway in isolated rat aorta

The impact of magnesium (Mg) on vascular tone is mainly due to its calcium antagonist activity resulting in smooth muscle relaxation. The present study was designed to determine the roles of nitric oxide and calcium ions in magnesium -induced vasorelaxation in rats aortic rings. In this study, the aorta contracted with NE (3x10-6M), pre-incubated with cyclooxygenase (COX) and epoxygenase inhibitors, then dose response curve(DRC) made to magnesium sulfate (MgSO4 ) served as a control. On the other hand, the aorta incubated with L-NAME (10 mM) ,nifedipine (10-5M) , indomethacin (10-5M) and clotrimazole (30 mM) for 20 minutes then contracted by nor-epinephrine (NE) and relaxed by MgSO4 doses. Interestingly, L-NAME shifted the DRC of MgSO4 to the left and elevated Emax significantly. Furthermore, p IC50 tended to increase in L-NAME pre-incubated aortic rings. Also, aortic rings that are pre-contracted with 10-6M NE in the absence of calcium ions shifted the DRC to the left but it significantly reduced pIC50. Furthermore, calcium channel blocker (Nifedipine) significantly reduced the relaxatory effects of magnesium ions and it markedly decreased Emax with altering pIC50 values significantly. The present results revealed that indomethacin pre-incubation slightly shifted the curve of MgSO4 to the right, while p IC50 was significantly increased and Emax did not significantly change in clotrimazole pre-incubation. In conclusions, the results of this study suggested that magnesium ions can probably relax aortic smooth muscles , and this is may be returned to in part to nitric oxide ,COX and epoxygenase enzyme pathways.

Diosmin, a Citrus Nutrient, Activates Imidazoline Receptors to Alleviate Blood Glucose and Lipids in Type 1-Like Diabetic Rats.

Diosmin is a nutrient that is widely contained in citrus and that has been indicated to improve glucose metabolism in diabetic disorders. Recently, we demonstrated that diosmin induces β-endorphin to lower hyperglycemia in diabetic rats. However, the mechanisms of diosmin in opioid secretion were unclear. Therefore, we focused on the secretion of opioids from isolated adrenal glands induced by diosmin. The changes in the released β-endorphin-like immunoreactivity (BER) were determined using ELISA. Diosmin increased the BER level in a dose-dependent manner, and this effect was markedly reduced in the absence of calcium ions. Activation of the imidazoline I-2 receptor (I-2R) has been introduced to induce opioid secretion. Interestingly, we observed that diosmin activates CHO cells expressing I-R. Additionally, diosmin-increased BER was inhibited by the blockade of I-2R in isolated adrenal glands. Additionally, an antagonist of I-2R blocked diosmin-induced effects, including the reduction in hyperglycemia and the increase in plasma BER in streptozotocin-induced diabetic rats (STZ-diabetic rats). Repeated treatment of STZ-diabetic rats with diosmin for one week induced changes in hepatic glycogen, lipid levels, and the expression of phosphoenolpyruvate carboxykinase (PEPCK). Furthermore, an antagonist of I-2R blocked the diosmin-induced changes. Additionally, plasma lipids modified by diosmin were also reversed by the blockade of I-2R in STZ-diabetic rats. Taken together, we suggest that diosmin may activate I-2R to enhance the secretion of β-endorphin from adrenal glands and to influence metabolic homeostasis, resulting in alleviation of blood glucose and lipids in STZ-diabetic rats.

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments.

Interaction of Daptomycin and Calcium with Lipid Bilayers

Daptomycin is a cyclic lipopeptide antibiotic recently approved for clinical treatment of gram positive bacterial infections. It displays strong bactericidal behavior in the presence of excess calcium ions and in biomembranes containing negatively charged PG headgroups. Yet, the molecular mechanism of daptomycin, beyond the suggestion of ion channel formation and cellular depolarization, remains unknown. Recent data has shown results inconsistent with ion channel formation, prompting a more detailed investigation of Daptomycin and calcium interactions with lipid bilayers at higher structural resolutions. In order to fully characterize the mechanism of Daptomycin, it is necessary to understand the role of individual components that are integral to Daptomycin's mode of action in the lipid bilayer. By using lamellar diffraction of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) multilayers, we examined the individual and combined effects of Daptomycin and calcium ions on bilayer structure. We found that calcium ions alone result in a slight thickening effect of the bilayer, partially supporting molecular simulations of calcium ions and PG bilayers that propose a calcium ordering effect at physiological calcium concentrations. Inclusion of daptomycin alone produced a bilayer thinning effect, suggesting that Daptomycin is capable of inserting into the headgroup region of the lipid bilayer during binding. Lastly, samples that contained both Daptomycin and calcium ions showed a bilayer thickening effect. These results suggest that Daptomycin binds to the membrane in the absence of calcium ions but induces a structural thickening of the bilayer in the presence of calcium ions. The apparent thickening effect of Daptomycin is unlike other membrane-active antibiotics, such as melittin or LL-37, which induce bilayer thinning. These studies may form the basis for more detailed investigations to characterize the molecular mechanisms of Daptomycin and other cyclic lipopeptides.

Calcium-independent binding of human C-reactive protein to lysophosphatidylcholine in supported planar phospholipid monolayers.

C-reactive protein (CRP), a major acute-phase pentraxin, binds to plasma membranes through the multivalent contacts with zwitterionic phosphorylcholine groups for activating classical complement systems. However, the interaction of CRP with phosphorylcholine-based biomaterials is unknown due to the lack of our understanding on the activation mechanism of CRP in altered local microenvironments. This paper reports the novel calcium-independent interaction of CRP to bioactive phospholipid lysophosphatidylcholine (LPC) in supported phospholipids monolayers as determined using SPR. Binding inhibition experiments indicate exposure of glycerophosphate moiety of LPC is responsible for the calcium-free interaction. Our study may explode the established concept that CRP requires calcium for binding to LPC on damaged cell membranes or biomaterials.

Real-Time Quartz Crystal Microbalance Monitoring of Free Docosahexaenoic Acid Interactions with Supported Lipid Bilayers.

Docosahexaenoic acid (DHA) is the most abundant polyunsaturated omega-3 fatty acid found in mammalian neuronal cell membranes. Although DHA is known to be important for neuronal cell survival, little is know about how DHA interacts with phospholipid bilayers. This study presents a detailed quartz crystal microbalance with dissipation monitoring (QCM-D) analysis of free DHA interactions with individual and mixed phospholipid supported lipid bilayers (SLB). DHA incorporation and subsequent changes to the SLBs viscoelastic properties were observed to be concentration-dependent, influenced by the phospholipid species, the headgroup charge, and the presence or absence of calcium ions. It was observed that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) SLBs incorporated the greatest amount of DHA concentration, whereas the presence of phospholipids, phosphatidylserine (PS), and phosphatidylinositol (PI) in a POPC SLB significantly reduced DHA incorporation and changed the SLBs physicochemical properties. These observations are hypothesized to be due to a substitution event occurring between DHA and phospholipid species. PS domain formation in POPC/PS 8:2 SLBs was observed in the presence of calcium ions, which favored DHA incorporation to a similar level as for a POPC only SLB. The changes in SLB thickness observed with different DHA concentrations are also presented. This work contributes to an understanding of the physical changes induced in a lipid bilayer as a consequence of its exposure to different DHA concentrations (from 50 to 200 μM). The capacity of DHA to influence the physical properties of SLBs indicates the potential for dietary DHA supplementation to cause changes in cellular membranes in vivo, with subsequent physiological consequences for cell function.