Biomedical subjects
Publications and source records attributed to M Glaser.
The definition of the chemical composition of human gallstones is shown by means of X-ray fine structure analysis. The simple method allowed a prompt distinction in stones poor and rich in cholesterol and of other ingredients. The method needs smallest test quantities (2 mg) in regard for a conservative medicamentous therapy.
Using multifrequency phase and modulation fluorometry and a nonlinear least-squares analysis of lifetime data, we were able to determine the complex decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in synthetic phospholipid bilayers. Our results showed a monoexponential decay of DPH in the pure isotropic solvents studied, over a wide temperature range, and a double-exponential decay of DPH in phospholipids, both above and below the transition. During the transition, and in mixed-phase phospholipids, a three-component analysis was successfully accomplished, and the pre-exponential factors of the two main components have been shown to be quantitatively representative of the gel and liquid-crystalline phases of the bilayer. The fractional intensity of the shorter lifetime component depends on the modalities of the sample preparation. The factors affecting this component are discussed. From the DPH fluorescence lifetime and from the anisotropy data in L-alpha-dimyristoyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidyl choline mixtures, a phase diagram was independently constructed. Conclusions about the sensitivity and the partition of the probe between gel and the liquid-crystalline phases of the bilayer are derived. Lifetime experiments on DPH in a L-alpha-dilauroyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidylch oline mixture suggested a general method for the determination and quantitation of the two different phases in the bilayer.
A fluorescence quenching method was developed for determining partition coefficients and diffusional rates of small molecules in cell membranes. This method involves quenching the fluorescence of carbazole-labeled membranes by hydrophobic molecules that partition into membranes. Cell membrane phospholipids of mouse LM cells in tissue culture were biosynthetically labeled with the carbazole moiety by supplementing the growth media with 11-(9-carbazolyl)undecanoic acid. Plasma membranes, microsomes, and mitochondria were isolated free of nonmembranous neutral lipids, and the incorporation of the fluorescent probe was characterized. Quenching studies of the carbazole moiety by a series of N-substituted picolinium perchlorate salts showed that the carbazole moiety was located in the hydrophobic interior of the membrane bilayer. The carbazole fluorescence also was quenched by the hydrophobic quenchers lindane, methoxychlor, and 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene, indicating that these compounds partitioned into the membrane. Stern-Volmer quenching constants determined by fluorescence lifetime and intensity measurements were identical, as expected for dynamic quenching. The effects of different lipid compositions on quenching constants and partition coefficients were determined by comparing different membrane fractions. These parameters also were measured in membranes from cells in which the phospholipid composition was altered by substituting ethanolamine for choline in the growth medium. Changes in the lipid composition produced changes in the bimolecular quenching constants. For example, bimolecular quenching constants for 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene were higher in mitochondrial membranes than in plasma membranes and microsomes. They were also higher in dispersions made from membrane phospholipids as compared with intact membranes or total lipid dispersion.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylate kinase prepared from a temperature-sensitive adk mutant of Escherichia coli was thermolabile at 40 degrees C while the wild type enzyme was stable. The degree of thermolability of the mutant enzyme was concentration-dependent in that a much greater thermolability was observed in the concentrated crude homogenate than in a 50-fold dilution of the crude homogenate. This concentration dependence was lost after extensive purification of the mutant adenylate kinase, although the enzyme was still thermolabile. A protein was identified that co-purified with the wild type and mutant enzyme through several purification steps and that altered the degree of thermolability of the mutant adenylate kinase. A homogeneous preparation of the adenylate kinase-associated protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel with Mr = 34,000. The interaction of this protein with adenylate kinase explains why the thermolability of the mutant adenylate kinase changed during purification and the dependence of the thermolability on concentration. The adenylate kinase-associated protein may be important in regulating the activity of adenylate kinase and subsequently affecting the rate of cell growth.
The phospholipid composition of primary rat hepatocytes was manipulated by supplementing the medium with choline analogues. The unnatural analogue l-2-amino-1-butanol was incorporated into membrane phospholipids to the largest extent, whereas the natural choline analogues ethanolamine, N-methylethanolamine, and N,N-dimethyl-ethanolamine were methylated to yield phosphatidylcholine. When cells were supplemented with [14C]ethanolamine, greater than 25% of the total phosphatidylcholine contained radiolabel in the polar head group after 2 days of supplementation. The extent of phospholipid methylation was reduced by depriving the cells of serine and methionine. Under these conditions, N-methylethanolamine and N,N-dimethylethanolamine were incorporated into phospholipids and were not further metabolized to phosphatidylcholine. After 3 days of supplementation with N-methylethanolamine, the content of phosphatidyl-methylethanolamine went from essentially 0 to 40% of the total phospholipids and surpassed the extent of incorporation of all other analogues. The formation of the new phospholipid species was primarily at the expense of phosphatidylcholine and phosphatidylethanolamine. D-beta-Hydroxybutyrate dehydrogenase, which requires phosphatidylcholine for activity, was assayed in submitochondrial membranes isolated from supplemented cells. For cells supplemented with either l-2-amino-1-butanol or N-methylethanolamine, the Km for NADH increased relative to choline-supplemented cells while the Km for acetoacetate remained the same. For example, after 3 days of supplementation with N-methylethanolamine, the Km for NADH was 3-fold higher than the value for the choline-supplemented control cells. The change in the Km was due to the change in the lipid environment with no alteration in the enzyme itself. The results suggest that the phosphatidylcholine molecules necessary to activate the enzyme exchange with the other phospholipids in the membrane so that the Km of the enzyme reflects the overall content of phosphatidylcholine as well as other properties of the membrane phospholipids.
Platelets incubated with prostaglandin synthetase inhibitors, i.e. aspirin (ASA) and diclofenac-sodium (Voltaren) show a decreased capability to aggregate. While ASA induces the complete loss of platelet pseudopodia, Voltaren causes the appearance of long, needle-shaped pseudopodia, a finding which may suggest a rather increased tendency to aggregate. Platelets labeled with ferritin hydrazide and examined with a transmission electron microscope and by X-ray analysis show a greater deposition of ferritin hydrazide after incubation with Voltaren in comparison to those incubated with ASA and to the controls. This finding suggests an increased negative charge of the stretched platelet membrane after incubation with Voltaren with a subsequent increase in the repulsive forces between platelets.
A fluorescence derivative of bovine cytochrome b5 was prepared by using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) in deoxycholate. Reaction conditions were established to specifically label the hydrophobic membrane-binding domain of the protein at a ratio of 0.9 +/- 0.1 dansyl group per cytochrome b5. Fluorescence measurements on the dansyl-labeled protein reflected the state of aggregation of the protein and its binding to lipids. The cytochrome b5 derivative was a sensitive probe for the detection of phospholipid phase transitions in reconstituted phospholipid vesicles. The rotational relaxation time of the labeled protein was strongly influenced by the phospholipid composition and the cholesterol content of the lipid bilayer, but it was largely insensitive to the integrity of the hydrophilic domain of the protein. When the membrane-binding domain of cytochrome b5 was bound to phospholipid vesicles, a preferential association with either the gel or the liquid-crystalline phase was not observed. The results suggest that the two domains of cytochrome b5 undergo predominantly independent motion and that the motion of the dansyl-labeled membrane-binding domain directly reflects the properties of the bulk lipids in the bilayer.
Whereas substantial evidence indicates that the majority of glioma patients make humoral immune responses to their own tumours, the evidence that glioma patients make significant cellular immune responses is more tenuous and controversial. In order to study those properties of human gliomas that might contribute to their ability to escape cell-mediated immune attack, we have examined the ability of cultured human glioma cells to elicit allogeneic cytolytic lymphocyte responses in vitro. Five of ten glioma lines were unable to elicit allogeneic cytolytic lymphocyte responses in mixed lymphocyte-tumour cultures, despite the presence of serologically detectable alloantigens on the surface of the glioma cells. Analysis of the reasons why certain glioma lines failed to stimulate cytolytic lymphocyte responses revealed three distinct mechanisms by which human gliomas may escape cellular immune attack: 1. a defect in immunogenicity which can be overcome by "help" from an allogeneic mixed lymphocyte reaction, 2. the secretion of a protective mucopolysaccharide coat, and 3. the production of macromolecular immunosuppressive substance(s). The implications of these findings for the immunotherapy of human gliomas are discussed.
For study of those properties of human gliomas that might contribute to their ability to escape cell-mediated immune attack, cultured human glioma cells were examined for their ability to elicit allogeneic cytolytic lymphocyte responses in vitro. Of 9 glioma lines, 5 were unable to elicit allogeneic cytolytic lymphocyte responses in mixed lymphocyte--tumor cultures although the concentration of stimulating glioma cells was varied over a fortyfold range. However, lymphocytes specifically cytolytic for 4 of the nonstimulatory lines could be generated if irradiated, third-party stimulator lymphocytes were added to cultures containing responder lymphocytes and glioma cells. The specific cytolytic lymphocytes produced in these cultures were inactivated by treatment with the monoclonal anti-T-cell antibody OKT3 plus complement and were thus identified as T-cells. However, nonspecific, non-T-lytic effectors were also generated. The results of these experiments demonstrated that certain cultured gliomas possessed a defect in immunogenicity that can be overcome by "help" from an allogeneic mixed lymphocyte reaction. The possible nature of this help and the potential implications of these results for the immunotherapy of human gliomas are discussed.
The effect of an electrostatic potential on the enzymatic activity of D-beta-hydroxybutyrate dehydrogenase was examined. Phospholipase D was used to increase the surface charge and concomitantly the electrostatic potential of submitochondrial membranes. The apparent Km for the negatively charged substrates of D-beta-hydroxybutyrate dehydrogenase increased as the membranes were reacted with phospholipase D. There was a 10-fold increase in the apparent Km for NADH when the content of acidic phospholipids was increased by 24%. The addition of monovalent or divalent cations, which reduced the electrostatic potential, largely reversed the apparent Km changes. At the same ionic strength, divalent cations had a substantially larger effect than monovalent cations. Similar results were obtained when the purified apoenzyme was reconstituted in unilamellar vesicles containing different ratios of phosphatidylcholine and acidic phospholipids. When the apoenzyme was reconstituted into phosphatidylcholine vesicles containing increasing amounts of phosphatidylethanolamine, the apparent Km also increased but to a smaller extent, and increasing the ionic strength did not reverse this effect. The results show that the apparent Km of D-beta-hydroxybutyrate dehydrogenase can be significantly altered by an electrostatic potential as well as other properties of the phospholipid polar head group.
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The origin of the envelope lipids acquired by Rous sarcoma virus (RSV) and vesicular stomatitis virus (VSV) during budding from the plasma membrane of chicken embryo fibroblasts was examined. Several differences were observed between the lipid composition of RSV and the plasma membrane. When the phospholipid composition of the cells was modified by growing them in the presence of the choline analogues, N,N-dimethylethanolamine or l-2-amino-1-butanol, the phospholipid composition of the virus was subsequently altered but in a very different manner than the plasma membrane. In the plasma membrane, the increase in the analogue-containing phospholipid was at the expense of phosphatidylcholine and phosphatidylethanolamine while the amount of sphingomyelin remained constant. In RSV, however, there was a decrease in sphingomyelin and phosphatidylethanolamine while there was only a small change in the amount of phosphatidylcholine. Phospholipid polar head group modification did not significantly alter the fatty acid composition or the cholesterol content. Membranes of phagosomes isolated after the cells had ingested latex beads had essentially the same phospholipid composition as the plasma membrane. The phospholipid composition of VSV was different from RSV, but it also did not reflect the composition of the plasma membrane. The composition of the plasma membrane was intermediate between the viruses and the endoplasmic reticulum, but contamination of the plasma membrane fraction with the endoplasmic reticulum could not account for the observed differences. These results show that the viruses bud from localized lipid regions that do not reflect the average properties of the plasma membrane.
Adenylate cyclase specific activities in membranes isolated from chicken embryo fibroblasts transformed by Rous sarcoma virus are significantly lower than the specific activity of the enzyme in normal membranes. Since normal and transformed membranes have different phospholipid and fatty acid compositions, adenylate cyclase activities were examined in normal and transformed membranes which had been supplemented with polar head groups or fatty acids. Basal, fluoride, and prostaglandin E1-stimulated activities changed systematically with phospholipid composition. Increases in the primary amino group of the phospholipid polar head groups or the average degree of fatty acid unsaturation both inhibited adenylate cyclase activity. In general, adenylate cyclase activities in normal membranes were more sensitive to phospholipid compositional changes compared to adenylate cyclase in transformed membranes. The data indicate that the lower adenylate cyclase activities in transformed membranes are not solely attributable to phospholipid changes but do suggest that increases in the percentage of phosphatidylethanolamine may contribute to the lower adenylate cyclase activities in transformed membranes.
The role of adherent phagocytic cells in an in vitro secondary cytotoxic response against Simian virus 40 (SV 40)-induced tumor-associated antigens was investigated. Spleen cells (responder cells), from mice primed with syngeneic SV 40-transformed cells, extensively depleted of macrophages by filtration through a Sephadex G-10 column followed by iron carbonyl treatment, had a markedly decreased capacity to generate in vitro secondary cytotoxic reactivity against syngeneic SV 40-transformed cells when cultured with the relevant stimulator cells. The secondary response was restored by the addition of adherent peritoneal cells from normal mice syngeneic to those immunized with the antigen. Within a certain dose range, small numbers of peritoneal cells completely reconstituted the response, whereas large numbers inhibited the reactivity. The restored cultures maintained specific cytotoxic reactivity against SV 40-induced tumor-associated antigens which was mediated by effector T cells as shown by sensitivity to anti-Thy-1.2 antiserum and complement. These results suggested a requirement for adherent phagocytic cells (accessory cells) in in vitro generation of a secondary, cytotoxic response to tumor-associated antigens.
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Complementation analysis with independently isolated plA and adk (adenylate kinase) mutants of Escherichia coli showed that all the mutants belong to the same complementation group. The results suggest that the adk (plsA) locus is the structural gene for adenylate kinase.
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