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Biological Membranes (biological + membrane)
Selected AbstractsPore-Forming Properties of Alamethicin F50/5 Inserted in a Biological MembraneCHEMISTRY & BIODIVERSITY, Issue 6 2007Natascia Vedovato Abstract The pore-forming properties of native and synthetic alamethicins were investigated in photoreceptor rod outer segments (OS) isolated from frog retina, and recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS within less than 50,ms by means of a computer-controlled micro-perfusion system. Once blocked with light, the main OS endogenous conductance, the OS membrane resistance was >1,G,, allowing low-noise and high-resolution recordings. Currents of ca. 700,pA were recorded in symmetric K+ (100,mM) and Ca2+ (1,mM), upon applying 1,,M of alamethicin F50/5 or its [L -Glu(OMe)7,18,19] analogue to the OS membrane (clamped at ,20,mV). In the latter peptide, the Gln residues at positions 7, 18, and 19 were substituted with side-chain esterified Glu residues. For both peptides, the current activated exponentially, with a delay from peptide application, and exponentially returned to zero without any delay, upon removing the peptide from the external solution. The delay as well as the activation (,a) and deactivation (,d) time constants of the current produced by the modified alamethicin were much slower, and the current noise was much larger, with respect to the corresponding values for alamethicin F50/5. Therefore, the above three Gln residues are not a key factor for pore formation, but the [L -Glu(OMe)7,18,19] analogue produces larger pores with a lower probability of formation. [source] Membrane proteins and membrane proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2008Sandra Tan Abstract Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins. [source] Membrane microdomains and proteomics: Lessons from tetraspanin microdomains and comparison with lipid raftsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2006François Le Naour Dr. Abstract Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures. [source] Cholesterol-rich membrane coatings for interaction studies in capillary electrophoresis: Application to red blood cell lipid extractsELECTROPHORESIS, Issue 20 2006Maria V. Lindén Abstract The purpose was to develop a stable biological membrane coating for CE useful for membrane interaction studies. The effect of cholesterol (chol) on the stability of dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin (SM) coatings was studied. In addition, a fused-silica capillary for CE was coated with human red blood cell (RBC) ghost lipids. Liposomes prepared of DPPC/SM with and without chol or RBC ghost lipids were flushed through the capillary and the stability of the coating was measured electrophoretically. Similar mixtures of DPPC/SM with and without chol were further studied by differential scanning calorimetry. The presence of phosphatidylcholine as a basic component in the coating solution of DPPC/SM/chol was found to be essential to achieve a good and stable coating. The results also confirmed the stability of coatings obtained with solutions of DPPC with 0,30,mol% of chol and SM in different ratios, which more closely resemble natural membranes. Finally, the electrophoretic measurements revealed that a stable coating is formed when capillaries are coated with liposomes of RBC ghost lipids. [source] Quantitative Determination of Surface Concentration of Human Apolipoprotein H with Capillary ElectrophoresisIUBMB LIFE, Issue 5 2000Shao-xiong Wang Abstract The phospholipid monolayer at an air/water interface is widely used to mimic the biological membrane. The dynamic process of the protein or peptide interacting with lipid molecules can be reflected in the change in surface pressure of the monolayer. But the conventional method used to measure the surface pressure change gives results that cannot easily be correlated with the contribution of a single protein molecule. Previously, measuring the surface concentration of the protein molecules at the air/water interface has required the protein to be labeled with radioactivity or fluorescence. Here, a new method using capillary electrophoresis is introduced to measure the surface concentration of the protein. The results show at least two advantages of the new method: The numerical results of protein concentration can be obtained in a more precise and rapid way; and there is no need to label the protein sample or to build a special monolayer setup. [source] Molecular dynamics simulation of the hydrocarbon region of a biomembrane using a reduced representation modelJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 14 2001Lewis Whitehead Abstract The development of a coarse-grained reduced-representation model of the hydrocarbon region of a biological membrane is reported. The potential is based on the popular Gay,Berne model of liquid crystals, and involves the linking of individual Gay,Berne ellipsoids by harmonic springs to form each hydrocarbon chain. Diffusion coefficients and order parameters have been calculated by molecular dynamics computer simulations for a range of parameter sets. The results clearly demonstrate the presence of a phase transition from an ordered low-temperature solid phase reminiscent of the L,, phase of phospholipids, to a high-temperature disordered phase reminiscent of the L, phase. Order parameters calculated for each layer of the model are consistent with the experimental segmental order parameters reported for dipalmitoyl phosphatidylcholine. The application of this model to the study of small molecule diffusion within the membrane core is proposed. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1622,1633, 2001 [source] The effect of phenyltin chlorides on osmotically induced erythrocyte haemolysisAPPLIED ORGANOMETALLIC CHEMISTRY, Issue 6 2005Adam Miszta Abstract The toxicity of many amphiphilic compounds may result from their effect on the lipid phase of biological membranes. Upon incorporation such compounds may change the properties of membranes in general and in particular alter the organization of membrane lipids. These changes should affect, among other things, the mechanical properties of membranes. We selected two amphiphilic compounds, diphenyltin dichloride (Ph2SnCl2) and triphenyltin chloride (Ph3SnCl), which are known to be located at different regions of the lipid bilayer and to be toxic. As a model biological membrane the erythrocyte plasma membrane was used. Analysis of the haemolysis kinetics showed differences between the effect of the compound studied on mechanical properties at so-called non-lytic concentrations. Diphenyltin dichloride showed a limited effect on erythrocyte haemolysis, whereas triphenyltin chloride affected all the parameters measured (extent of initial haemolysis, extent of final haemolysis and membrane mechanical strength). We correlated these effects with the location of the investigated compounds in liposomes. The presented data show that triphenyltin chloride reduces the erythrocyte plasma membrane mechanical strength and increases the extent of haemolysis under osmotic stress conditions. Copyright © 2005 John Wiley & Sons, Ltd. [source] Channel-like crystal structure of cinchoninium L - O -phosphoserine salt dihydrateCHIRALITY, Issue 6 2010Aleksandra Wese, ucha-Birczy Abstract Studies on the interactions between L - O - phosphoserine, as one of the simplest fragments of membrane components, and the Cinchona alkaloid cinchonine, in the crystalline state were performed. Cinchoninium L - O -phosposerine salt dihydrate (PhSerCin) crystallizes in a monoclinic crystal system, space group P21, with unit cell parameters: a = 8.45400(10) Å, b = 7.17100(10) Å, c = 20.7760(4) Å, , = 90°, , = 98.7830(10)°, , = 90°, Z = 2. The asymmetric unit consists of the cinchoninium cation linked by hydrogen bonds to a phosphoserine anion and two water molecules. Intermolecular hydrogen bonds connecting phosphoserine anions via water molecules form chains extended along the b axis. Two such chains symmetrically related by twofold screw axis create a "channel." On both sides of this channel cinchonine cations are attached by hydrogen bonds in which the atoms N1, O12, and water molecules participate. This arrangement mimics the system of bilayer biological membrane. Chirality 2010. © 2009 Wiley-Liss, Inc. [source] Effects of methylcyclodextrin on lysosomesFEBS JOURNAL, Issue 5 2001Michel Jadot The cholesterol complexing agent methyl-cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl- l -phenylalanine 2-naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications. [source] Bioaccumulation Assessment Using Predictive Approaches,INTEGRATED ENVIRONMENTAL ASSESSMENT AND MANAGEMENT, Issue 4 2009John W Nichols Abstract Mandated efforts to assess chemicals for their potential to bioaccumulate within the environment are increasingly moving into the realm of data inadequacy. Consequently, there is an increasing reliance on predictive tools to complete regulatory requirements in a timely and cost-effective manner. The kinetic processes of absorption, distribution, metabolism, and elimination (ADME) determine the extent to which chemicals accumulate in fish and other biota. Current mathematical models of bioaccumulation implicitly or explicitly consider these ADME processes, but there is a lack of data needed to specify critical model input parameters. This is particularly true for compounds that are metabolized, exhibit restricted diffusion across biological membranes, or do not partition simply to tissue lipid. Here we discuss the potential of in vitro test systems to provide needed data for bioaccumulation modeling efforts. Recent studies demonstrate the utility of these systems and provide a "proof of concept" for the prediction models. Computational methods that predict ADME processes from an evaluation of chemical structure are also described. Most regulatory agencies perform bioaccumulation assessments using a weight-of-evidence approach. A strategy is presented for incorporating predictive methods into this approach. To implement this strategy it is important to understand the "domain of applicability" of both in vitro and structure-based approaches, and the context in which they are applied. [source] On the efficiency of cell-like and tissue-like recognizing membrane systemsINTERNATIONAL JOURNAL OF INTELLIGENT SYSTEMS, Issue 7 2009Miguel A. Gutiérrez-Naranjo Cell-like recognizing membrane systems are computational devices in the framework of membrane computing inspired from the structure of living cells, where biological membranes are arranged hierarchically. In this paper tissue-like recognizing membrane systems are presented. The idea is to consider that membranes are placed in the nodes of a graph, mimicking the cell intercommunication in tissues. In this context, polynomial complexity classes associated with recognizing membrane systems can be defined. We recall the definition for cell-like systems, and we introduce the corresponding complexity classes for the tissue-like case. Moreover, in this paper two efficient solutions to the satisfiability problem are analyzed and compared from a complexity point of view. © 2009 Wiley Periodicals, Inc. [source] Imbalance of plasma membrane ion leak and pump relationship as a new aetiological basis of certain disease statesJOURNAL OF INTERNAL MEDICINE, Issue 6 2003G. Ronquist Abstract. The basis for life is the ability of the cell to maintain ion gradients across biological membranes. Such gradients are created by specific membrane-bound ion pumps [adenosine triphosphatases (ATPases)]. According to physicochemical rules passive forces equilibrate (dissipate) ion gradients. The cholesterol/phospholipid ratio of the membrane and the degree of saturation of phospholipid fatty acids are important factors for membrane molecular order and herewith a determinant of the degree of non-specific membrane leakiness. Other operative principles, i.e. specific ion channels can be opened and closed according to mechanisms that are specific to the cell. Certain compounds called ionophores can be integrated in the plasma membrane and permit specific inorganic ions to pass. Irrespective of which mechanism ions leak across the plasma membrane the homeostasis may be kept by increasing ion pumping (ATPase activity) in an attempt to restore the physiological ion gradient. The energy source for this work seems to be glycolytically derived ATP formation. Thus an increase in ion pumping is reflected by increased ATP hydrolysis and rate of glycolysis. This can be measured as an accumulation of breakdown products of ATP and end-products of anaerobic glycolysis (lactate). In certain disease entities, the balance between ATP formation and ion pumping may be disordered resulting in a decrease in inter alia (i.a.) cellular energy charge, and an increase in lactate formation and catabolites of adenylates. Cardiac syndrome X is proposed to be due to an excessive leakage of potassium ions, leading to electrocardiographic (ECG) changes, abnormal Tl-scintigraphy of the heart and anginal pain (induced by adenosine). Cocksackie B3 infections, a common agent in myocarditis might also induce an ionophore-like effect. Moreover, Alzheimer's disease is characterized by the formation of extracellular amyloid deposits in the brain of patients. Perturbation of cellular membranes by the amyloid peptide during the development of Alzheimer's disease is one of several mechanisms proposed to account for the toxicity of this peptide on neuronal membranes. We have studied the effects of the peptide and fragments thereof on 45Ca2+ -uptake in human erythrocytes and the energetic consequences. Treatment of erythrocytes with the ,1,40 peptide, results in qualitatively similar nucleotide pattern and decrease of energy charge as the treatment with Ca2+ -ionophore A23187. Finally, in recent studies we have revealed and published in this journal that a rare condition, Tarui's disease or glycogenosis type VII, primarily associated with a defect M-subunit of phosphofructokinase, demonstrates as a cophenomenon an increased leak of Ca2+ into erythrocytes. [source] The methylmercury- l -cysteine conjugate is a substrate for the L-type large neutral amino acid transporterJOURNAL OF NEUROCHEMISTRY, Issue 4 2008Zhaobao Yin Abstract Methylmercury (MeHg) is a potent neurotoxin. The mechanism(s) that governs MeHg transport across the blood-brain barrier and other biological membranes remains unclear. This study addressed the role of the L-type large neutral amino acid transporter, LAT1, in MeHg transport. Studies were carried out in CHO-k1 cells. Over-expression of LAT1 in these cells was associated with enhanced uptake of [14C]-MeHg when treated with l -cysteine, but not with the d -cysteine conjugate. In the presence of excess l -methionine, a substrate for LAT1, l -cysteine-conjugated [14C]-MeHg uptake was significantly attenuated. Treatment of LAT-1 over-expressing CHO-k1 cells with l -cysteine-conjugated MeHg was also associated with increased leakage of lactate dehydrogenase into the media as well as reduced cell viability measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. In contrast, knock-down of LAT1 decreased the uptake of l -cysteine-conjugated MeHg and attenuated the effects of MeHg on lactate dehydrogenase leakage and CHO-k1 cell viability. These results indicate that the MeHg- l -cysteine conjugate is a substrate for the neutral amino acid transporter, LAT1, which actively transports MeHg across membranes. [source] Antimicrobial peptide interactions with silica bead supported bilayers and E. coli: buforin II, magainin II, and arenicin,JOURNAL OF PEPTIDE SCIENCE, Issue 8 2009Ryan W. Davis Abstract Using the unique quantitative capabilities of hyperspectral confocal microscopy combined with multivariate curve resolution, a comparative approach was employed to gain a deeper understanding of the different types of interactions of antimicrobial peptides (AMPs) with biological membranes and cellular compartments. This approach allowed direct comparison of the dynamics and local effects of buforin II, magainin II, and arenicin with nanoporous silica bead supported bilayers and living E. coli. Correlating between experiments and comparing these responses have yielded several important discoveries for pursuing the underlying biophysics of bacteriocidal specificity and the connection between structure and function in various cellular environments. First, a novel fluorescence method for direct comparison of a model and living system is demonstrated by utilizing the membrane partitioning and environmental sensitivity of propidium iodide. Second, measurements are presented comparing the temporal dynamics and local equilibrium concentrations of the different antimicrobial agents in the membrane and internal matrix of the described systems. Finally, we discuss how the data lead to a deeper understanding of the roles of membrane penetration and permeabilization in the action of these AMPs. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Interaction of S413 -PV cell penetrating peptide with model membranes: relevance to peptide translocation across biological membranesJOURNAL OF PEPTIDE SCIENCE, Issue 5 2007Miguel Mano Abstract Cell penetrating peptides (CPPs) have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest both in vitro and in vivo, although the mechanisms by which the cellular uptake occurs remain unclear and controversial. Following our previous work demonstrating that the cellular uptake of the S413 -PV CPP occurs mainly through an endocytosis-independent mechanism, we performed a detailed biophysical characterization of the interaction of this peptide with model membranes. We demonstrate that the interactions of the S413 -PV peptide with membranes are essentially of electrostatic nature. As a consequence of its interaction with negatively charged model membranes, the S413 -PV peptide becomes buried into the lipid bilayer, which occurs concomitantly with significant peptide conformational changes that are consistent with the formation of a helical structure. Comparative studies using two related peptides demonstrate that the conformational changes and the extent of cell penetration are dependent on the peptide sequence, indicating that the helical structure acquired by the S413 -PV peptide is relevant for its nonendocytic uptake. Overall, our data suggest that the cellular uptake of the S413 -PV CPP is a consequence of its direct translocation through cell membranes, following conformational changes induced by peptide-membrane interactions. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] The role of surfactants in the reversal of active transport mediated by multidrug resistance proteinsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2003Katrijn Bogman Abstract A variety of seven nonionic, one amphoteric and, one anionic surfactant that are applied or investigated as surfactants in drug formulation, were analyzed for their capacity to modulate carrier-mediated transport by efflux pumps. Two cell lines, murine monocytic leukemia cells overexpressing P-glycoprotein (P-gp) and Madin-Darby canine kidney cells stably overexpresssing human multidrug resistance-associated protein 2 (MRP2), were used as test systems. The modulation of P-gp and of MRP2 function was studied by the reversal of rhodamine 123 and of methylfluorescein-glutathione conjugate transport, respectively. Mechanisms that were not transporter related and could lead to misinterpretations were identified, such as probe quenching, probe encapsulation by micelles, and membrane damage. P-gp-mediated rhodamine 123 transport was inhibited by five nonionic surfactants in a concentration-dependent manner and in the order TPGS,>,Pluronic PE8100,>,Cremophor EL,>,Pluronic PE6100,,,Tween 80. In contrast, none of the surfactants showed a significant inhibition of MRP2-mediated efflux in Madin-Darby canine kidney/MRP2 cells. In conclusion, the results indicate that surfactants demonstrate a transporter-specific interaction, rather than unspecific membrane permeabilization. The present analysis offers insight in the possible mechanisms of surfactant interactions with biological membranes and could help to identify specific drug formulations. © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1250,1261, 2003 [source] Design, synthesis and properties of novel iron(III)-specific fluorescent probesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 4 2004Wei Luo ABSTRACT Bidentate chelators such as hydroxypyridinones and hydroxypyranones are highly iron selective. The synthesis of two novel fluorescent probes N -[2-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-2-(7-methoxy-2-oxo-2H -chromen-4-yl)acetamide (CP600) and N -[(3-hydroxy-6-methyl-4-oxo-4H -pyran-2-yl)methyl]-2-(7-methoxy-2-oxo-2H -chromen-4-yl)acetamide (CP610) is reported. The method involves coupling the bidentate ligands, 3-hydroxypyridin-4-one and 3-hydroxypyran-4-one, with the well-characterised fluorescent probe methoxycoumarin. Fluorescence emission of both probes at 380 nm is readily quenched by Fe3+. The fluorescence was quenched to a greater extent by Fe3+ than by Mn2+, Co2+, Zn2+, Ca2+, Mg2+, Na+ and K+ and to approximately the same extent as Cu2+. Comparison of the fluorescence-quenching ability by a range of metal ions on CP600 and CP610 and the hexadentate chelator, calcein, under in-vitro conditions, demonstrated advantages of the two novel fluorescent probes with respect to both iron(III) sensitivity and selectivity. Chelation of iron(III) by CP600 and CP610 leads to the formation of a complex with a metal-to-ligand ratio of 1:3. Fluorescence is quenched on formation of such complexes. These probes possess a molecular weight less than 400 and thus they are predicted to permeate biological membranes by passive diffusion, and have potential for reporting intracellular organelle labile iron levels. [source] In-vitro and in-vivo studies of cefpirom using bile salts as absorption enhancersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2003Yahya Mrestani ABSTRACT Cephalosporins have to be administered by injection because of the poor intestinal absorption of the orally delivered drugs. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefpirom (Cp) is a new semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin that has been substituted in position 3 with a cyclopenteno-pyridinium group in order to create a zwitterionic compound. It exhibits highly hydrophilic properties, as shown from its extremely low partition coefficient, and therefore its lipophilicity was increased using bile salts. The effect of this on the partition coefficients determined in the n-octanol/buffer system was confirmed using an in-vitro transport model with artificial and biological membranes. The pharmacokinetic properties of Cp were investigated in rabbits after intraduodenal administration with and without bile salts. Furthermore, the physiological compatibility of the bile salts was investigated using active D-glucose transport. [source] Poly(L-lysine) as a model drug macromolecule with which to investigate secondary structure and membrane transport, part I: physicochemical and stability studiesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 3 2002Montakarn Chittchang Low oral bioavailability of therapeutic peptides and proteins generally results from their poor permeability through biological membranes and enzymatic degradation in the gastrointestinal tract. Since different secondary structures exhibit different physicochemical properties such as hydrophobicity, size and shape, changing the secondary structure of a therapeutic polypeptide may be another approach to increasing its membrane permeation. Poly(L-lysine) was used as a model polypeptide. The objectives of this study were to induce secondary structural changes in poly(L-lysine) and to determine the time course over which a given conformer was retained. In addition, the hydrophobicity of each secondary structure of poly(L-lysine) was assessed. The circular dichroism (CD) studies demonstrated that the conditions employed could successfully induce the desired secondary structural changes in poly(L-lysine). The ,-helix conformer appeared to be more stable at 25° C whereas the ,-sheet conformer could be preserved at 37° C. On the other hand, the random coil conformer was retained at both temperatures. Significant losses of the ,-helix and the ,-sheet conformers were observed when the pH was reduced. The change in ionic strength did not affect any of the conformers. The octanol/buffer partitioning studies indicated that the ,-helix and the ,-sheet conformers exhibited significantly different (P< 0.05) hydrophobicities. In conclusion, variation of pH and temperature conditions can be used to induce secondary structural changes in poly(L-lysine). These changes are reversible when the stimuli are removed. The ,-helix and the ,-sheet conformers of poly(L-lysine) are more lipophilic than the native random coil conformer. Thus, poly(L-lysine) may represent an ideal model polypeptide with which to further investigate the effects of secondary structure on membrane diffusion or permeation. [source] Immunological Detection of in Vitro Formed Phosphatidylethanol,An Alcohol Biomarker,With Monoclonal AntibodiesALCOHOLISM, Issue 6 2008Antti E. Nissinen Background:, Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography,mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. Methods:, C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. Results:, We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. Conclusions:, The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes. [source] Ischemia,reperfusion injury pathophysiology, part IJOURNAL OF VETERINARY EMERGENCY AND CRITICAL CARE, Issue 4 2004DACVECC, Maureen McMichael DVM Abstract Objective: To review the current scientific literature on ischemia,reperfusion (IR) injury in both human and veterinary medicine. To describe the normal antioxidant defense mechanisms, the pathophysiology of IR injury, and the role of neutrophils in IR injury. Data sources: Data sources include scientific reviews and original research publications in both human and veterinary medicine. Summary: IR injury is a complex pathophysiological process involving numerous pathways and body systems. Normal antioxidant defense mechanisms function to limit oxidative injury during times of health. Ischemia is the period that occurs before oxygenated blood is re-introduced and the severity of injury has been shown to correlate with the magnitude and length of ischemia in dogs. During ischemia, there is a buildup of substances (i.e., xanthine oxidase, hypoxanthine, etc.) that, upon re-introduction of oxygen, form reactive oxygen species (ROS). ROS, produced in large part upon reperfusion, can cause extensive damage to DNA, proteins, carbohydrates, and lipids. Although mammalian systems are endowed with abundant antioxidant defenses, the generation of large amounts of ROS can overwhelm these mechanisms leading to cell dysfunction and death. Neutrophils play a critical role in IR injury and may mediate the majority of mucosal and microvascular injury that occurs by releasing ROS and proteolytic enzymes. Although experimental studies have been carried out on cats, dogs, and horses there are few clinical studies on companion animals. Conclusions: The pathophysiology of IR injury is complex and involves damage by ROS to all biological membranes. Neutrophils play a major role in IR injury and initiate and propogate much of the damage. This article is intended as a review of the pathophysiology of IR injury. [source] Ischemia,reperfusion injury: assessment and treatment, part IIJOURNAL OF VETERINARY EMERGENCY AND CRITICAL CARE, Issue 4 2004DACVECC, Maureen McMichael DVM Abstract Objective: To review the current scientific literature on ischemia,reperfusion (IR) injury in both human and veterinary medicine and to describe the assessment of IR injury, the available testing methods, and the options available for treatment. Data sources: Data sources include scientific reviews and original research publications in both human and veterinary medicine. Summary: The assessment of IR injury includes measuring products formed by the reaction of reactive oxygen species (ROS) with biological membranes, measuring levels of endogenous antioxidants, and measuring ROS themselves. Testing depends on the laboratory used, the test method chosen, the sample submitted (i.e., plasma, urine, tissue, etc.), and the timing of the test in relation to sample collection. For this reason, testing is not standardized and pharmacological data on antioxidant effectiveness are not available. Antioxidants and drugs tested have included single agents as well as ,cocktails' consisting of several agents working at different key points in the injury cascade. Conclusions: There are several new testing methods as well as new strategies for attempting to ameliorate the damage inflicted upon reperfusion and this article is intended as a review of the assessment and treatment of IR injury. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 8 2009The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive). Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 1 2009The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive). Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 11-12 2008The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive). Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 11-12 2007The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive). Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 1 2007The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive). Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 12 2006The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive. Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 11 2006The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive. Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] Cover Picture: QSAR Comb.MOLECULAR INFORMATICS, Issue 10 2006The transmembrane protein aquaporin spans biological membranes and forms channels that allow the passage of water molecules. The water channels consist of alpha helices (purple cylinders) and are shown as a Conolly surface,in which the electrostatic potential is represented by colors (blue: negative, red: positive. Peter Agre and Roderick MacKinnon were awarded the 2003 Nobel Prize in chemistry for the discovery of water channels and for structural and mechanistic studies on ion channels, respectively. Cover illustration by courtesy of Prof. Dr. Jürgen Brickmann, Dr. Thorsten Borosch, MOLNET e.V. [source] | ||||||||||||||||||||||||||||