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Biomedical subjects

M Glaser

Publications and source records attributed to M Glaser.

At least 37 records · Page 2Linked to original sources
PubMed1998

Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: an electrostatic model and experimental results.

Direct fluorescence digital imaging microscopy observations demonstrate that a basic peptide corresponding to the effector region of the myristoylated alanine-rich C kinase substrate (MARCKS) self-assembles into membrane domains enriched in the acidic phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). We show here that pentalysine, which corresponds to the first five residues of the MARCKS effector region peptide and binds to membranes through electrostatic interactions, also forms domains enriched in PS and PIP2. We present a simple model of domain formation that represents the decrease in the free energy of the system as the sum of two contributions: the free energy of mixing of neutral and acidic lipids and the electrostatic free energy. The first contribution is always positive and opposes domain formation, whereas the second contribution may become negative and, at low ionic strength, overcome the first contribution. Our model, based on Gouy-Chapman-Stern theory, makes four predictions: 1) multivalent basic ligands, for which the membrane binding is a steep function of the mole fraction of acidic lipid, form domains enriched in acidic lipids; domains break up at high concentrations of either 2) basic ligand or 3) monovalent salt; and 4) if multivalent anionic lipids (e.g., PIP2) are present in trace concentrations in the membrane, they partition strongly into the domains. These predictions agree qualitatively with experimental data obtained with pentalysine and spermine, another basic ligand.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

Apprehension about communication and human resilience.

This study examined the relationship between scores on two personality characteristics, apprehension about communication and resilience, from 200 elementary school teachers from several counties who completed the Personal Report of Communication Apprehension and the Personal Resilience Questionnaire. Scores on the two measures correlated -.33, which accounts for 10% of the common variance.

Adaptation, Psychological↗
PubMed1997

Measurement of the rate of myelination using a fluorescent analogue of ceramide.

Fluorescence digital imaging microscopy was used to investigate the process of myelin formation by Schwann cells in neuronal cocultures. The uptake of the fluorescent ceramide analogue N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]D-erythro-sphingosine (C5-DMB-ceramide) and its return to the plasma membrane as the corresponding fluorescent sphingomyelin and galactocerebroside analogues were measured. Through observation of this process it was possible to determine the rate of lipid synthesis in myelin internodes. The highest rate of synthesis of fluorescent sphingomyelin and galactocerebroside analogues was observed between days 3 and 7 after induction of myelination. This rate was approximately 5-fold greater than the steady-state rate of synthesis in fully myelinated internodes and 10-fold higher than the rate observed prior to myelination. The internode diameter increased during the first 3 days of myelination, but this was followed by a reduction in diameter and then an increase until the myelin sheath formation was completed. Internodes were found to be heterogeneous in terms of lipid distribution, with fluorescence intensities ranging 5-fold in myelinating cultures. Additionally, the rate of lipid transport along the internode was slow since there was a quicker increase in fluorescence intensity near the cell body of the Schwann cell than near the nodes of Ranvier. The results show that fluorescence digital imaging microscopy can be used to study the process of myelin formation and to determine the rate of formation, lipid transport, and heterogeneity of the myelin membrane.

Animals↗
PubMed1997

Membrane penetration depth and lipid phase preference of acyl-labeled dansyl phosphatidylcholines in phosphatidylcholine vesicles.

The dansyl fluors of 1-oleoyl-2-[4-(dansyl)amino]butyl]-sn-glycero-3-phosphocholine (18:1,4-dansyl PC) and 1-palmitoyl-2-[1-[(dansyl)amino)]undecanoyl]-sn-glycero-3-phosphoc holine (16:0,11-dansyl PC) were shown to reside at similar depths in phosphatidylcholine vesicles at pH 7. Analysis of fluorescence emission maxima showed that the dansyl groups of both 18:1,4-dansyl PC and 16:0,11-dansyl PC in phosphatidylcholine vesicles experienced environments more polar than methanol, suggesting that the dansyl group attached to the terminus of the undecanoyl chain must fold back toward the bilayer surface. Fluorescence polarization measurements in solid/fluid lipid mixtures show that both probes partition strongly into fluid phase lipid. The very low polarization values of 16:0,11-dansyl PC in lipid vesicles are consistent with the notion that the dansyl fluor of 16:0,11-dansyl PC existed in an environment allowing a high degree of motional freedom due to folding back of the dansyl group attached to the undecanoyl chain.

Dansyl Compounds↗
PubMed1996

Formation of membrane domains during the activation of protein kinase C.

The lateral membrane organization of phosphatidylserine, diacylglycerol, substrate, and Ca(2+)-dependent protein kinase C in large unilamellar vesicles was investigated by using fluorescence digital imaging microscopy. The formation of phosphatidylserine domains could be induced by either Ca2+, the MARCKS peptide, or protein kinase C. However, only Ca2+ could induce diacylglycerol to partition into the phosphatidylserine domains. In the complete protein kinase C assay mixture, two separate triple-labeling experiments demonstrated the colocalization of phosphatidylserine, protein kinase C, diacylglycerol, and the MARCKS peptide in domains. The amounts of all the labeled components in whole vesicles and in domains were measured at various concentrations of either phosphatidylserine, Ca2+, diacylglycerol, or the MARCKS peptide or with the addition of polylysine. The role of each component in forming membrane domains and in mediating the enzyme activity was analyzed. The results indicated that the inclusion of the MARCKS peptide in the domains, not just the binding of the substrate to vesicles, was especially important for PKC activity. The formation of PKC domains required the presence of DAG and Ca2+ at physiological ionic strength. The PKC activity was proportional to the amounts of PKC and substrate in the domains. The results also showed that the MARCKS peptide left the domains after being phosphorylated. A model for the activation of protein kinase C involving sequestering of the reaction components into membrane domains is proposed. The efficiency of the reaction was greatly increased by concentrating the activators, the enzyme, and the substrate into domains.

Amino Acid Sequence↗
PubMed1996

Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.

Animals↗
PubMed1996

Substrate binding causes movement in the ATP binding domain of Escherichia coli adenylate kinase.

Crystallographic evidence suggests that there is a large hinged domain motion associated with substrate binding in adenylate kinase. To test this hypothesis, resonance energy transfer measurements of substrate binding were initiated. Adenylate kinase from Escherichia coli consists of three domains: the main body of the enzyme with alpha-helical and beta-sheet secondary structure, and domains that close over the AMP and ATP binding sites. Four single tryptophan mutants were constructed to map distances. Two tryptophan mutants were positioned at residues 133 (Y133W) and 137 (F137W), which are in the domain that closes over the ATP binding site. Mutant F86W that is located at the AMP binding site, and mutant S41W that is in the loop that close over AMP, complete the mapping library. Energy transfer was measured between each of these tryptophans and 5-[[2-(acetylamino)ethyl]amino]naphthalene-1-sulfonic acid (AEDANS) covalently bound to the single cysteine residue at position 77, which is located in the main body of adenylate kinase. The distance between the tryptophan of the F137W mutant adenylate kinase and the AEDANS-labeled Cys-77 decreased by 12.1 A upon the binding of the bisubstrate inhibitor P1, P5-bis(5'-adenosyl) pentaphosphate (AP5A). There were only small alterations in the tryptophan to Cys-77-AEDANS distances in the Y133W, F86W, and S41W mutants upon the binding of AP5A, ATP, or AMP, implying that movement of residues 133, 86, and 41 in relation to the Cys-77 residue was minimal. These results suggest that there is significant closure of the ATP binding domain upon the binding of ATP or AP5A. Unexpectedly, exposure of the enzyme to AMP also introduced a partial closure of the ATP hinged domain.

Adenosine Triphosphate↗
PubMed1995

Formation of membrane domains created during the budding of vesicular stomatitis virus. A model for selective lipid and protein sorting in biological membranes.

Vesicular stomatitis virus buds from domains of the plasma membrane that have a unique protein and lipid composition. Fluorescence digital imaging microscopy and resonance energy transfer were used to determine how the two viral envelope-associated proteins, the G and the M proteins, could alter the lateral distribution of lipids in large unilamellar vesicles and form domains. The G protein formed large domains in vesicles containing phosphatidic acid but not with phosphatidylserine, while the M protein formed domains enriched in both acidic phospholipids. Domains enriched in sphingomyelin were observed only when both the G protein and the M protein were present in vesicles containing phosphatidic acid. Phosphatidylcholine and gramicidin (chosen to represent a host membrane protein) were excluded from the domains. Cholesterol was induced to partition into the domains only in vesicles containing phosphatidic acid and sphingomyelin along with both of the proteins. Phosphatidylethanolamine was not enriched or depleted in the domains. Domains of similar composition were formed using vesicles made from dioleoylphospholipids and the lipids extracted from BHK-21 plasma membranes, indicating that the fatty acid composition was not as important as the polar head groups of the phospholipids. The phospholipid and cholesterol compositions of the domains formed by the G and the M proteins in vesicles were very similar to the composition of the viral envelope, suggesting that the domains represent the areas in the plasma membrane where the virus buds. This study provides a model for selective lipid and protein sorting that occurs in biological membranes.

Animals↗
PubMed1995

Membrane domains containing phosphatidylserine and substrate can be important for the activation of protein kinase C.

The relationship between lipid domains and enzyme activity was studied via the direct visualization and quantitation of domains by fluorescence digital imaging microscopy. The substrate used in these experiments was a basic peptide derived from a prominent cellular substrate (MARCKS) of protein kinase C. The MARCKS peptide and phosphatidylserine, which were labeled by two different fluorophores, colocalized into domains in large vesicles (5-10 microns). Increasing the ionic strength disrupted the domains of the MARCKS peptide and phosphatidylserine, and this was accompanied by a decrease in protein kinase C activity. Dansylpolylysine, which inhibits protein kinase C, was similar to the MARCKS peptide in forming domains enriched in phosphatidylserine. The degree of enrichment of the MARCKS peptide in the phosphatidylserine domains decreased proportionally with protein kinase C activity when polylysine was added. Polylysine caused the MARCKS peptide to be displaced from the domains into the nondomain areas of the vesicles. This suggested that binding of the substrate to the vesicles was not the critical factor for protein kinase C activity, but rather it was the organization of the substrate into domains that was related to the activation of the enzyme. Gramicidin, which was chosen to represent a neutral membrane protein, was excluded from the domains with phosphatidylserine, and it had no effect on the enrichment of the domains or the enzyme activity. The results of this study show that the formation of membrane domains can be important for the activation of protein kinase C and the activity can be inhibited by disrupting the domains.

Amino Acid Sequence↗
PubMed1994

Lipid domains in model and biological membranes.

Lipid domains that occur within biological of model membranes encompass a variety of structures with very different lifetimes. The separation of membrane lipids into compositional domains can be due to lateral phase separation, immiscibility within a single phase, or interaction of lipids with integral or peripheral proteins. Lipid domains can affect the extent and rate of reactions in the membrane and provide sites for the activity of specialized proteins. Domains are likely to be involved in the process of lipid sorting to various cellular membranes, as well as in other processes which involve membrane budding or invagination.

Animals↗
PubMed1994

Formation of membrane domains by the envelope proteins of vesicular stomatitis virus.

The properties of the two envelope-associated proteins of vesicular stomatitis virus, the glycoprotein (G) and the matrix protein (M), were investigated in order to understand the mechanism of virus budding and domain formation in membranes. Fluorescence resonance energy transfer was used to study the interaction between the G protein and specific phospholipids. The protein had the highest affinity for phosphatidic acid among the phospholipids tested. Fluorescence digital imaging microscopy also was used to determine how the protein could alter the lateral distribution of phospholipids in membranes. Large domains enriched in phosphatidic acid were observed when the protein was incorporated into phospholipid vesicles. The G protein colocalized with the phosphatidic acid-enriched domains. Similar experiments carried out with the M protein showed that the M protein induced the formation of domains enriched not only in phosphatidic acid but also in phosphatidylserine. The phosphatidic acid-enriched domains induced by either the G or M proteins were similar in terms of the degree of enrichment of phosphatidic acid and the size of the domains. When the two proteins were reconstituted in vesicles at the same time, the domains were condensed. There was a greater degree of phosphatidic acid enrichment, and the size of the domains was reduced. The formation of domains enriched in the viral proteins and specific phospholipids may mimic the first steps that occur during budding of the virus from the plasma membrane of infected cells.

Animals↗
PubMed1994

The closed conformation of a highly flexible protein: the structure of E. coli adenylate kinase with bound AMP and AMPPNP.

The structure of E. coli adenylate kinase with bound AMP and AMPPNP at 2.0 A resolution is presented. The protein crystallizes in space group C2 with two molecules in the asymmetric unit, and has been refined to an R factor of 20.1% and an Rfree of 31.6%. In the present structure, the protein is in the closed (globular) form with the large flexible lid domain covering the AMPPNP molecule. Within the protein, AMP and AMPPNP, and ATP analog, occupy the AMP and ATP sites respectively, which had been suggested by the most recent crystal structure of E. coli adenylate kinase with Ap5A bound (Müller and Schulz, 1992, ref. 1) and prior fluorescence studies (Liang et al., 1991, ref. 2). The binding of substrates and the positions of the active site residues are compared between the present structure and the E. coli adenylate kinase/Ap5A structure. We failed to detect a peak in the density map corresponding to the Mg2+ ion which is required for catalysis, and its absence has been attributed to the use of ammonium sulfate in the crystallization solution. Finally, a comparison is made between the present structure and the structure of the heavy chain of muscle myosin.

Adenosine Monophosphate↗
PubMed1994

"Grandma, please"!

An intergenerational telephone reassurance line for latchkey children helps them cope with being home alone and keeps the volunteer older adults in the mainstream.

Aged↗
PubMed1993

Distributions of proteins and lipids in the erythrocyte membrane.

Fluorescence digital imaging microscopy was used to characterize erythrocyte membrane domains. To investigate the role of specific proteins in forming lipid domains, erythrocyte ghosts were double-labeled with fluorescent phospholipids and a fluorescent label specific for band 3, the major integral membrane protein. Areas of enrichment, or domains, were observed for both the protein and lipid components. The size and enrichment of the protein and lipid domains in the membrane depended upon the ionic strength but not the temperature. Also, there was a higher correlation between the areas of band 3 and phosphatidylcholine enrichment than between band 3 and phosphatidylserine enrichment. This is consistent with band 3 playing a role in the formation of the phosphatidylcholine domains. Furthermore, areas of spectrin enrichment also were observed in immunofluorescently labeled ghosts, and the band 3 domains were found to coincide with the spectrin domains. Thus, the distribution of band 3 in ghosts is influenced by interactions with the cytoskeleton. Together, these experiments illustrate the type of membrane heterogeneity present in erythrocyte ghosts and the type of lipid and protein interactions that can affect the domains.

Animals↗
PubMed1993

Pyrularia thionin binding to and the role of tryptophan-8 in the enhancement of phosphatidylserine domains in erythrocyte membranes.

Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that addition of the peptide to erythrocyte membranes as well as to vesicles formed from phospholipids isolated from erythrocyte membranes causes an enhancement of phospholipid domains which are made visible by the use of fluorescence digital imaging microscopy with fluorescent derivatives of PS (NBD-PS) and phosphatidylcholine (NBD-PC). Addition of thionin caused a large increase in NBD-PS domains, with an accompanying enrichment of NBD-PC in another separate domain. Double-labeling experiments performed with a Texas Red derivative of thionin show that the peptide binds to the domain enriched in NBD-PS. P thionin inactivated by modification of Trp-8 with N-bromosuccinimide lost the ability to enhance PS domains, although it bound to the membrane with the same affinity as native P thionin. This shows that binding to the membrane is not in itself sufficient to cause the NBD-PS and NBD-PC redistribution into domains.

Animals↗
PubMed1993

Brachytherapy for recurrent nasopharyngeal and naso-ethmoidal tumours.

Recurrent or persistent carcinoma of the nasopharynx or naso-ethmoid region occurs even after combined surgery and radical external irradiation. The confined bony access and proximity of vital structures may compromise resection margins. Brachytherapy using implantable radioactive gold or iodine seeds is a useful adjunct to the skull base surgeon in eliminating residual microscopic disease. We report nine cases with recurrent or persistent disease of the nasopharynx or naso-ethmoid regions treated with brachytherapy with encouraging results.

Adolescent↗
PubMed1992

Heparin-associated thrombocytopenia: successful therapy with the heparinoid Org 10172 in a patient showing cross-reaction to LMW heparins.

A patient suffering from heparin-associated thrombocytopenia (HAT), recurrent arteriothromboses, and acute renal failure after treatment with standard heparin is described. He failed to improve when therapy was continued with low-molecular-weight (LMW) heparin (Fragmin, Kabi Pfrimmer, Erlangen, FRG). By means of the in vitro heparin-induced platelet activation (HIPA) assay it was shown that standard heparin and the LMW heparins Fragmin and Fraxiparin (Sanofi Labaz, Munich, FRG), as well as the enoxaparine Clexane (Nattermann, Cologne, FRG), all induced platelet activation with the patient's serum. In contrast, the LMW heparinoid Org 10172 (Organon, Oss, The Netherlands) did not cause platelet activation. When the patient was subsequently treated by parenteral administration of Org 10172 as anticoagulant over a period of several weeks the number of platelets rapidly increased and the patient almost completely recovered. This case shows that strong in vivo and in vitro cross-reactivity between standard heparin and LMW heparins may occur, but can be avoided by the use of a novel heparinoid, Org 10172. The HIPA assay provides a simple and sensitive laboratory method for the choice of an innocuous heparin or heparinoid for continued parenteral anticoagulation.

Aged↗