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M Glaser

Publications and source records attributed to M Glaser.

At least 55 records · Page 3Linked to original sources
PubMed1992

Effects of hydrostatic pressure on a membrane-enveloped virus: high immunogenicity of the pressure-inactivated virus.

A new approach to the preparation of antiviral vaccines relying on the inactivation of the virus particle by hydrostatic pressure is described. The enveloped virus vesicular stomatitis virus was utilized as a model; a pressure of 260 MPa applied for 12 h reduced infectivity by a factor of 10(4), and the antibodies against pressurized material were as effective as those against the intact virus when measured by their neutralization titer. Fluorescence measurements indicate that application of pressure results in perturbations of the particle interactions that permit binding of specific molecular probes. Electron microscopy showed that the membrane of the pressurized virus was partially preserved, presenting the spike pattern of the membrane G protein. Unlike the icosahedral viruses, dissociation into smaller particles was not observed, but a constant change in the morphology was the presence of a bulge in the surface of the pressurized virus, indicating a displacement of the capsid subunits, retained under the lipid and protein membrane.

Animals↗
PubMed1991

Orientational distribution of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles as determined by global analysis of frequency domain fluorimetry data.

Polarized differential phase and modulation ratios were obtained for 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles by using multifrequency phase fluorometry. Data were analyzed in terms of both empirical sums-of-exponentials modeling and directly in terms of the orientational distribution functions. The orientational analysis model was used to recover the angular distribution of DPH and the rotational diffusion coefficient in the various membrane model systems throughout the phase transition. A global analysis methodology was utilized to obtain an internally consistent set of parameters that fit all of the data simultaneously. The rank order parameters (P2) and (P4) were extracted from the experimental data, and the angular distribution functions of DPH were calculated. When the time-zero anisotropy (r0) of several sets of data taken at various temperatures were linked in a single global analysis, better recovery of the fourth rank order parameter (P4), diffusion constant D, and r0 was obtained with respect to the unlinked analysis. From these recovered values, a detailed picture concerning the orientational distribution of DPH in membranes as a function of temperature was obtained. The results suggest that a single population of DPH molecules was present in the bilayers with their orientational distributions dependent upon the physical state of the membranes in the pure phases. During the phase transition, a superposition of two populations corresponding to the population of the pure phases was present. As the temperature increased in the transition region, one population was increasing at the expense of the other.

Diffusion↗
PubMed1991

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.

4-Chloro-7-nitrobenzofurazan↗
PubMed1991

Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase.

Site-directed mutagenesis of key amino acids of adenylate kinase has been used to suggest a new model for the location of the AMP and ATP binding sites. Phe-86 and Tyr-133, which are in close contact with the inhibitor Ap5A according to previous crystallographic results, have been independently changed to tryptophan and other amino acids. The Phe-86----Trp mutant had a 3- to 6-fold change in the Km for ATP and a 44-fold increase in the Km for AMP with a simultaneous loss of AMP substrate inhibition. Thus Phe-86 is probably in close contact with bound AMP. The Tyr-133----Trp mutant showed no large effects on enzyme kinetics and suggests that the previous assignment of Ap5A occupying natural adenosine binding sites is probably incorrect. A temperature-sensitive Leu-107----Gln mutant showed a 6-fold decrease in the Km for ATP and no effect on AMP binding, suggesting that this amino acid is near the ATP binding site. Changes in the fluorescence of single tryptophan-containing mutant enzymes provided specific information about AMP and ATP binding. The fluorescence results are consistent with the kinetic studies, and also suggest that AMP substrate inhibition is caused by the formation of an abortive complex that prevents the release of product.

Adenosine Monophosphate↗
PubMed1990

Antibodies against the cuticlin of Ascaris suum cross-react with epicuticular structures of filarial parasites.

The insoluble cuticlin from the cortical zone of the cuticle of adult Ascaris suum was purified and used to raise antibodies in C57/bl mice. The specificity of the antibodies for the external cortical layer was shown in the indirect immunofluorescence antibody test and by immunoelectronmicroscopy. A very high specificity for the external cortical layer was found. Some cross-reactions with cuticular collagens occurred, and increased after booster immunizations. The anti-cuticlin antibody cross-reacted with the electron-dense layers of the cortical zones of adult Acanthocheilonema viteae and Brugia pahangi. A very weak reaction was found in the cortical zone of adult Onchocerca volvulus. In the cuticles of third stage larvae of all three species mainly epitopes in the cortical zones were labelled. In no case did the anti-cuticlin antibody interact with the outermost surface of the cuticle.

Animals↗
PubMed1989

Efficient cloning of a mutant adenylate-kinase-encoding gene from Escherichia coli.

An optimized system has been developed for the transfer of a mutant gene from the Escherichia coli chromosome to a plasmid carrying the wild type (wt) allele. The wt allele was first cloned into a low-copy-number, self-transmissible plasmid with a single EcoRI, HindIII, and BamHI site. The plasmid was then transferred to a mutant strain that had been previously transformed with a high-copy-number plasmid carrying the recA+ gene to allow efficient homologous recombination. A 15% frequency of homogenotization was obtained during cloning of an adk gene that encodes a temperature-sensitive adenylate kinase (AK). The mutant AK had decreased mobility on sodium dodecyl sulfate-polyacrylamide gels compared with the wt enzyme. This was due to a point mutation that changed leucine-107 in the wt enzyme to glutamine-107 in the mutant enzyme as determined by nucleotide sequencing.

Adenylate Kinase↗
PubMed1989

Influence of proteins on the reorganization of phospholipid bilayers into large domains.

Using large (5-10 microns) vesicles formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera, and digital image processor, we examined the effects of membrane proteins on phospholipid domain formation. In vesicles composed of phosphatidic acid and phosphatidylcholine, incubation with cytochrome c induced the reorganization of phospholipids into large phosphatidic acid-enriched domains with the exclusion of phosphatidylcholine. Cytochrome c binding was demonstrated to be highest in the phosphatidic acid-enriched domain of the vesicle using the absorbance of the heme moiety for visualization. Both binding of cytochrome c and phospholipid reorganization were blocked by pretreatment of the vesicles with 0.1 M NaCl. The pore forming peptide gramicidin was examined for the effects of an integral protein on domain formation. Initially, gramicidin distributed randomly within the vesicle and showed no phospholipid specificity. Phosphatidic acid domain formation in the presence of 2.0 mM CaCl2 or 100 microM cytochrome c was not affected by the presence of 5 mol % gramicidin within the vesicles. In both cases, gramicidin was preferentially excluded from the phosphatidic acid-enriched domain and became associated with phosphatidylcholine-enriched areas of the vesicle. Thus, cytochrome c caused a major reorganization of both the phospholipids and the proteins in the bilayer.

Cytochrome c Group↗
PubMed1989

Effectiveness of triazolam, diazepam, and placebo as preanesthetic medications.

Eighty-three ASA Physical Status 1-2 patients were orally premedicated with triazolam (0.125, 0.25, or 0.5 mg), diazepam (5, 10, or 15 mg), or placebo to evaluate the effectiveness of these drugs and doses in reducing preoperative anxiety, providing sedation, and producing amnesia. The drug treatments were administered in a randomized, double-blind manner. The results obtained with each drug (dose) group were compared against those of the placebo group as a control. Changes in anxiety at 60 min after drug administration were evaluated: 1) by a trained anesthesia nurse clinician using an analog scale, 2) by the patient using the same analog scale, and 3) by the patient with the Multiple Affect Adjective Check List (MAACL). Changes in sedation at 60 min were also evaluated by the patient and nurse clinician using an analog scale. Amnesia was assessed by postoperative recall of picture cards shown to the patient 1 h after receiving preanesthetic medication. There were no significant differences between any drug (dose) and placebo for changes in patient-evaluated anxiety or sedation on the analog scale. With the other measures of anxiety, only triazolam (0.5 mg) reduced anxiety more than did placebo on both the patient (MAACL) and the nurse (analog) scales. With the nurse (analog) measure of sedation, only the highest doses of triazolam and diazepam were more sedating than placebo. Triazolam (0.5 mg) was the only drug dose that produced significant amnesia. The authors conclude that drug effects on anxiety, sedation, or amnesia that are statistically significant versus placebo effect are seen at only the highest doses of triazolam (0.5 mg) and diazepam (15 mg).

Adult↗
PubMed1988

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene reveal the effect of cholesterol on the microheterogeneity of erythrocyte membrane.

The fluorescence decay of 1,6 diphenyl-1,3,5-hexatriene (DPH) has been used to characterize aspects of the erythrocyte membrane structure related to the microheterogeneity of the lipid bilayer. The DPH decay has been studied using frequency domain fluorometry and the data analyzed either by a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values. The main intensity fraction was associated with a lifetime value centered at about 11 ns in the erythrocyte membrane, but a short component of very low fractional intensity had to be considered to obtain a good fit to the data. The lifetime value of the long component was insensitive to temperature, while the width of the distribution decreased with increasing temperature. In multilamellar liposomes prepared from phospholipids extracted from the erythrocytes, the long lifetime component showed a temperature dependence. The depletion of 27% of the cholesterol in the erythrocyte membrane induced a broadening of the distribution, suggesting a homogenizing effect of cholesterol. This effect has also been detected in egg phosphatidylcholine at a very low cholesterol/phospholipid molar ratio. The role of cholesterol on membrane heterogeneity is discussed in relation to the effect of cholesterol on water penetration.

Cell Fractionation↗
PubMed1988

Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer.

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.

Dansyl Compounds↗
PubMed1988

Visualization of domain formation in the inner and outer leaflets of a phospholipid bilayer.

Large vesicles (5-10-micron in diameter) were formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera and digital image processor. When such vesicles contained a fluorescent phosphatidic acid (PA) and were exposed to 2 mM CaCl2 or 0.5 mM PrCl3, it was possible to visualize PA-enriched domains within the vesicles. Calcium-induced domain formation was reversible in the presence of 4 mM EGTA. Vesicles were formed containing fluorescent PA on either the inner or outer leaflet of the bilayer and the patching and dissolution of patching were studied under conditions where calcium was present on the outside of the vesicle and where calcium was distributed across the bilayer. In addition, vesicles were formed with two different fluorescent PA's, one on the inner leaflet and a different one on the outer leaflet of the bilayer. The results of the experiments show that in vesicles formed primarily with naturally occurring phospholipids such as egg phosphatidylcholine or brain phosphatidylethanolamine, there was no coordinate action of the two leaflets of the bilayer. An exception to this was found, however, if the vesicles were formed in the presence of primarily dioleoyl phospholipids (greater than 95 mol %). In these vesicles there was a coordinate or coupled response to calcium by the two leaflets of the bilayer. In most cases, however, the two leaflets of the bilayer showed independent or uncoupled domain formation.

Calcium↗
PubMed1987

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles.

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Surgery, chemotherapy and whole abdominal radiotherapy in the management of advanced ovarian carcinoma.

A pilot study on 35 women was performed to determine the feasibility of giving whole abdominal radiotherapy after surgery and chemotherapy for Stage III and IV ovarian adenocarcinoma. The planned duration of therapy was only 6 months from diagnosis. Chemotherapy consisted of four courses of cis-platinum 100 mg/m2 alternating every 10-12 days with four courses of methotrexate 300 mg/m2 and cyclophosphamide 500 mg/m2 followed by folinic acid rescue. Twenty-nine of 35 (83%) patients were shown at second look surgery after chemotherapy to have residual tumour deposits no more than 2 cm in diameter. The usual radiotherapy dose was 2400 cGy in 20 fractions over approximately 4 to 5 weeks, with kidney shielding, and was generally well tolerated. Three patients failed to complete radiotherapy, two because of progressive disease and one because of persistent nausea and vomiting. During radiotherapy the white blood count fell below 2.0 X 10(9)/litre in seven patients (26%), and in eight patients (30%) the platelet count fell below 75 X 10(9)/litre. However, the white blood count nadir of 1.5 X 10(9)/litre was reached by 1300 cGy and the platelet nadir of 45 X 10(9)/litre was reached by 1400 cGy and both then levelled or recovered despite continuing radiotherapy. The median follow up since diagnosis is 26 months. Four of 12 patients with no tumour detected at second look operation have relapsed compared with 13 of 15 patients who had detectable tumour. Toxicity of this multi-modality therapy was acceptable.

Abdomen↗
PubMed1987

Visualization of Ca2+-induced phospholipid domains.

Large vesicles (5-15 microns) were formed by hydrating a dried lipid film containing phospholipids labeled with a fluorophore in one fatty acid chain. By using a fluorescence microscope attached to a low-light-intensity charge-coupled-device camera and digital-image processor, the vesicles were easily viewed and initially showed uniform fluorescence intensity across the surface. The fluorescence pattern of vesicles made with a fluorophore attached to phosphatidylcholine or phosphatidylethanolamine was unaffected by the presence of divalent cations such as Ca2+, Mg2+, Mn2+, Zn2+, or Cd2+. The fluorescence pattern of vesicles containing a fluorophore attached to the acidic phospholipids phosphatidylserine or phosphatidic acid showed distinct differences when treated with Ca2+ or Cd2+, although they were unaffected by Mg2+, Mn2+, or Zn2+. Treatment with 2.0 mM Ca2+ or Cd2+ resulted in the movement of the fluorophore to a single large patch on the surface of the vesicle. When vesicles were formed in the presence of 33 mol % cholesterol, patching was seen at a slightly lower Ca2+ concentration (1.0 mM). The possibility of interactions between Ca2+ and acidic phospholipids in plasma membranes was investigated by labeling erythrocytes and erythrocyte ghosts with fluorescent phosphatidic acid. When Ca2+ was added, multiple (five or six) small patches were seen per individual cell. The same pattern was observed when vesicles formed from whole lipid extracts of erythrocytes were labeled with fluorescent phosphatidic acid and then treated with Ca2+. This shows that the size and distribution of the Ca2+-induced domains depend on phospholipid composition.

Animals↗
PubMed1985

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.

Biophysical Phenomena↗
PubMed1985

[Fine-structure roentgen analysis for the determination of gallstone components].

Results of the determination of the chemical composition of human gallstones using X-ray diffraction method are reported. With simple handling it is possible beside identification of some other components to distinguish stones rich or poor in cholesterol already for smallest amounts of sample (greater than or equal to 10 mg, for instance endoscopically obtained material). This is significant with respect to the conservative medicamental therapy of lysis.

Calcium↗