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Biomedical subjects

R Blumenthal

Publications and source records attributed to R Blumenthal.

At least 163 records · Page 9Linked to original sources
PubMed1981

Interaction of tubulin with phospholipid vesicles. I. Association with vesicles at the phase transition.

Purified soluble tubulin will interact with highly sonicated small unilamellar vesicles of dipalmitoyl phosphatidylcholine. This results in the leakage of a fluorescent dye, carboxyfluorescein, from the internal aqueous space of the vesicles at the lipid phase transition. The amount of dye leaked from the vesicles is determined by the tubulin to vesicle ratio and partial dye leakage reflects the uniform leakage of carboxyfluorescein from all of the vesicles. Once tubulin interacts with dipalmitoyl phosphatidylcholine vesicles at the phase transition, it is unavailable to interact with additional vesicles. This interaction represents the formation of stable tubulin-vesicle recombinants as shown by density gradient centrifugation. Such recombinants are only formed by interacting the protein with the vesicles at the lipid phase transition temperature. The interaction is insensitive to ionic strength (0.001 to 4 M NaCl) and the recombinants remain stable for days.

Animals↗
PubMed1981

Interaction of tubulin with phospholipid vesicles. II. Physical changes of the protein.

We have shown that soluble tubulin will bind to small unilamellar vesicles of dipalmitoyl phosphatidylcholine (Klausner, R. D., Kumar, N., Weinstein, J. N., Blumenthal, R., and Flavin, M. (1981) J. Biol. Chem. 256, 5879-5885). This association uniquely occurs at the lipid phase transition. The tubulin, when bound to the vesicles, displays an altered tryptophan fluorescence characterized by a 5-nm blue shift in the emission maximum and a 22% decrease in fluorescence intensity, when compared to soluble tubulin. Tryptophans in vesicle-bound tubulin are less accessible to the aqueous collisional quenchers, acrylamide and iodide, than in soluble tubulin. Circular dichroism studies reveal an increase in alpha-helical content of tubulin as a result of vesicle interaction. Proteolytic digestion by trypsin of vesicle bound tubulin is slower than of soluble tubulin. The beta subunit of tubulin is preferentially protected from trypsin by vesicle interaction. Furthermore, the pattern of tryptic cleavage products is altered by this interaction.

Acrylamides↗
PubMed1981

Diagnosing borderline personality. A pilot study using multiple diagnostic methods.

The research described in this paper stemmed from the hypothesis that borderline personality organization can be differentiated from neurotic and psychotic levels of personality organization by means of three structural criteria: degree of identity integration, level of defensive operations, and capacity for reality testing. In order to elicit these criteria, the "structural" interview has been developed that focuses on the "here-and-now" patient-interviewer interaction. The patient's responses to the interviewer's attempts to clarify, confront, and interpret various aspects of the patient's interview behavior provide the basis for judgments as to the patient's structural diagnosis. Specifically, the paper reports a study of the differential diagnosis of 48 hospitalized patients in which structural diagnoses of borderline or psychotic personality organization were made according to this diagnostic interview approach. These diagnoses were compared with ones obtained from Gunderson's Diagnostic Interview for Borderlines, with psychological test diagnoses, and with clinical diagnoses based on past history and current illness. Results show substantial convergent agreement among all of the diagnostic methods and support the utility of the structural interview. In most discrepant cases, other methods reflected disagreement among themselves despite the diagnoses obtained from the structural interview, suggesting that there are some cases difficult to classify by any means. Further analysis suggests that the structural interview may be eliciting a different dimension of personality functioning in arriving at borderline diagnoses than do the other methods studied. The results also indicate that borderline structural diagnoses refer to patients described clinically as having severe character pathology, and do not overlap with patients described as having schizophrenic disorders. The structural interview appears to warrant further study, and, at the same time, shows promise as a research tool in further studies of structural diagnosis and its relevance for prognosis and treatment.

Adolescent↗
PubMed1980

Voltage-dependent translocation of the asialoglycoprotein receptor across lipid membranes.

A membrane receptor protein for asialoglycoproteins induces voltage-dependent increases in ion conductance across a lipid bilayer, probably reflecting penetration of the protein into the bilayer towards an electrically positive pole. In the presence of specific ligand for the receptor, this penetration leads to a 'translocation' of the receptor from one side of the bilayer to the other. These observations suggest a mechanism by which biological membranes might regulate the disposition of their proteins, and a way in which membrane receptors involved in endocytosis might be spared lysosomal destruction in order to be recycled to the plasma membrane.

Animals↗
PubMed1980

Lysophosphatidylcholine in liposomal membranes: enhanced permeability but little effect on transfer of a water-soluble fluorescent marker into human lymphocytes.

In an attempt to enhance delivery of liposome contents into cells, we tested the effect of lysophosphatidylcholine on transfer of the fluorescent dye, carboxyfluorescein, from small unilamellar and large multilamellar vesicles to human lymphocytes. Dioleoyl phosphatidylcholine and dioleoyl phosphatidylcholine-lysophosphatidylcholine small unilamellar vesicles with varying lipid ratios were prepared and characterized. In the presence of lysophosphatidylcholine, small unilamellar vesicles were slightly smaller and more leaky than those made without lysophosphatidylcholine. Lysophosphatidylcholine induced less leakage in large multilamellar vesicles. It did not show any appreciable effect on transfer of liposome contents, whether included as part of the liposomal bilayer (of unilamellar or multilamellar vesicles) or added exogenously together with small unilamellar dioleoyl phosphatidylcholine vesicles.

Cell Membrane Permeability↗
PubMed1980

Thin-layer chromatography with agarose gels. A quick, simple method for evaluating liposome size.

Thin-layer gels can be made with agarose and used to assess within a few minutes the efficiency with which multilamellar vesicles are converted to small unilamellar ones by sonication. A fluroescent lipid marker or vesicle-encapsulated solute permits continuous monitoring of the chromatography. Advantages over agarose gel column chromatography include speed of analysis, small sample size, the possibility of running multiple samples and simultaneously, and direct accessibility to fluorescence microscopy. This approach should also be useful in the study of liposome-lipoprotein interactions and in affinity chromatography of liposomes.

Chromatography, Agarose↗
PubMed1980

Receptor-mediated endocytosis of antibody-opsonized liposomes by tumor cells.

Specific receptor-mediated delivery of the contents of small, sonicated liposomes was studied with three murine tumor cell types: an IgG Fc receptor-negative nonphagocytic line (EL4); an Fc receptor-positive phagocytic line (P388D1); and an Fc receptor-positive nonphagocytic line (P388). The liposomes (formed from phosphatidylcholines, cholesterol, and dinitrophenyl-substituted phosphatidylethanolamine) contained carboxyfluorescein as a fluorescent marker and methotrexate as a pharmacologic agent. Binding and internalization of the liposomes were observed by fluorescence microscopy and measured by flow microfluorometry. The hapten-derivatized lipid was used as a binding point on the liposome for the antibody-combining site of the immunoglobulin. In the presence of IgG anti-dinitrophenyl, but not F(ab')2 or IgA anti-dinitrophenyl, liposomes bound to the Fc receptor-bearing cells. The liposomes underwent endocytosis by the P388D1 cells and, to a lesser extent, by the P388 cells. As measured by depression of [3H]deoxyuridine incorporation, methotrexate in IgG-opsonized liposomes had a much greater pharmacologic effect on the P388D1 cells than did the same amount in unopsonized liposomes or in free solution. This observation indicates that an appropriately chosen drug, incorporated in liposomes, can exert its effect on a cytoplasmic target after endocytosis. P388 cells showed a moderate effect of the drug in liposomes. Neither P388 nor P388D1 cells bound or ingested unopsonized liposomes, and the Fc receptor-negative EL4 line neither bound nor ingested opsonized liposomes. The data demonstrate specific interaction of opsonized liposomes with the cells' IgG Fc receptor.

Animals↗
PubMed1980

Reconstitution of the hepatic asialoglycoprotein receptor with phospholipid vesicles.

A solubilized detergent-free preparation of the hepatic binding protein specific for asialoglycoproteins associates spontaneously with small unilamellar lipid vesicles. This process is independent of the phase transition of the lipid and effectively restores the specific binding activity of the receptor protein. The insensitivity of the resulting lipid-protein complex to ionic strength provides evidence for a hydrophobic interaction. There is a perturbation of the lipid phase transition concomitant with addition of the protein. Circular dichroism studies indicate that the protein undergoes a conformational change on association with lipid. Binding of specific ligand produces further physical changes in the receptor as indicated by alterations in the tryptophan fluorescence quenching pattern.

Animals↗
PubMed1979

Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes.

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.

Animals↗
PubMed1979

Binding of antigen-bearing fluorescent liposomes to the murine myeloma tumor MOPC 315.

Small unilamellar lipid vesicles bearing the DNP-hapten on their surfaces and containing the water-soluble fluorescent dye carboxyfluorescein were formed by sonication. These vesicles were incubated with cells from the murine myeloma tumor MOPC 315, which secrete and also bear on the cell surface an immunoglobulin with affinity for the nitrophenyl hapten. At 0 degrees C the cells bound an average of several thousand vesicles at saturation. This binding was specific for the nitrophenyl hapten on the vesicle since it was abolished by an excess of soluble nitrophenyl derivative, by omission of the hapten from the vesicle, or by substitution for MOPC 315 of a tumor lacking receptors for the nitrophenyl hapten. Specific binding of vesicles was greater when cells were incubated at 37 degrees C. The study suggests that ligand-bearing vesicles can be a useful marker for cell surface immunoglobulin. However, in spite of the ability to "target" vesicles to cell surface determinants, binding did not result in increased delivery of vesicle contents to the cytoplasm.

Animals↗
PubMed1978

Design of liposomes for enhanced local release of drugs by hyperthermia.

Liposomes can be designed to release an entrapped drug preferentially at temperatures attainable by mild local hyperthermia. In a test system in vitro, protein synthesis by Escherichia coli is inhibited and killing of the cells is enhanced by heating neomycin-containing liposomes to their phase transition temperature to maximize drug release. In the presence of serum the ratio of release at 44 degrees C to that at 37 degrees C can be made greater than 100:1, suggesting possible applications in the treatment of tumors or local infection.

Escherichia coli↗
PubMed1978

Antibody-mediated targeting of liposomes. Binding to lymphocytes does not ensure incorporation of vesicle contents into the cells.

Small sonicated lipid vesicles containing the water-souble fluorescent dye 6-carboxyfluorescein were formed from dioleoyl phosphatidylcholine and the antigenic lipid N-dinitrophenylaminocaproyl phosphatidylethanolamine. When these vesicles were incubated with trinitrophenyl-modified human lymphocytes and divalent anti-trinitrophenyl antibody, the antibody bound 5000 to 15 000 vesicles to each cell. Binding was detected by fluorescence microscopy and quantitated by fluorometry and flow microfluorometry. Binding was three times greater with F(ab')2 fragments than with the whole antibody and, as expected, was almost absent with the monovalent F(ab') fragments. It was also absent or greatly reduced, (i) with control immunoglobulin G, (ii) in the presence of excess soluble trintrophenyl hapten, or (iii) if hapten was omitted from either cells or vesicles. It was unaffected by sodium azide and 2-deoxy-D-glucose but was markedly decreased at 3 degrees C. It was not reversed by incubation at 3 degrees C with excess trinitrophenyl lysine. Self-quenching of the fluorescence of 6-carboxyfluorescein was used to distinguish between release of vesicle contents into the cells and simple binding of intact vesicles (Weinstein, J.N., Yoshikami, S., Henkart, P., Blumenthal, R. and Gagins, W.A. (1977) Science 195, 489--491). Antibody-mediated binding led to little or no increase over spontaneous background levels in the amount of vesicle contents released into the lymphocytes.

Antibodies↗
PubMed1978

Dopamine-beta-hydroxylase-induced changes in the electrical conductance of bimolecular lipid membranes.

Dopamine-beta-hydroxylase (DBH), an enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE) in adrenal medullary chromaffin granules, increases the electrical conductance of bimolecular lipid membranes. The conductances increase requires both DA and Ca2+ and occurs in discrete steps. The conductance, which increases as the square of the DBH concentration, is nonselective for cations over anions and requires the native conformation of DBH. NE cannot replace DA.

Adrenal Glands↗
PubMed1977

Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker.

When small, unilamellar lipid vesicles containing a high concentration of the fluorescent dye 6-carboxyfluorescein are incubated with either frog retinas or human lymphocytes, fluroescence distributes widely throughout each cell. Since "self-quenching" largely prevents the dye from fluorescing as long as it remains sequestered in vesicles, it is clear that a considerable amount of dye is released from the vesicles and diluted into the much larger volume of the cell.

Animals↗