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M Smolarsky

Publications and source records attributed to M Smolarsky.

15 recordsLinked to original sources
PubMed1986

A novel lipid-conjugated marker for the direct labeling of the schistosomulum membrane.

Introduction of the fluorescent group lissamine rhodamine B into the surface of schistosomula of S. mansoni was achieved by brief incubation of the worms with liposomes carrying the lipid bound fluorophore in their bilayers. The liposomes were made of egg lecithin (PC) and lissamine-phosphatidylethanolamine (lissamine-PE). The lissamine groups could be directly detected on the parasite membrane by observation of the single worms under a fluorescent microscope. The fluorescent marker was employed to measure the lateral diffusion coefficient (D) of lipids in the schistosomular membrane and as a surface marker during the isolation of the schistosomula surface membrane.

Animals↗
PubMed1984

Acetylcholinesterase of Schistosoma mansoni: antigenic cross-reactivity with Electrophorus electricus and its functional implications.

Acetylcholinesterase (AChE) of the parasite Schistosoma mansoni cross-reacts with rabbit antibodies against AChE from the electric eel. Cross-reactivity was demonstrated by radioimmunoassay with sonicate, as well as soluble, preparations from various stages of the parasite life cycle. On sucrose density gradient, the parasite enzyme migrates as an 7.5 S form, whereas an 9.5 S peak is observed with the enzyme-antibody complex. Immunofluorescence microscopy shows specific staining of intact schistosomula as well as of adult worms. Moreover, the interaction of these antibodies resulted in a marked complement-dependent cytotoxicity towards intact schistosomula, indicating that AChE is an antigen of significance on the surface of the parasite.

Acetylcholinesterase↗
PubMed1984

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni.

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.

Acetylcholinesterase↗
PubMed1983

A method of introducing lipid-conjugated antigens into the surface of schistosomula.

Introduction of synthetic antigens into the surface of schistosomula of Schistosoma mansoni was achieved by brief incubation of the worms with liposomes carrying the lipid-bound antigens in their bilayers. Three-hour-old schistosomula were surface-labeled with lipid-conjugated dinitrophenyl (DNP) groups by using liposomes made of egg lecithin-N-dinitrophenil-epsilon-aminocaproyl-phosphatidylethanolamine (5:1). The DNP groups incorporated in this way could be detected for more than 21 hours in vitro by using rabbit anti-DNP antibodies stained with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Immunofluorescence microscopy showed the lipid antigen to be uniformly distributed over the entire surface of the worms. Electron microscope studies, performed with purified rabbit anti-DNP antibodies followed by ferritin-conjugated goat anti-rabbit IgG, showed that the DNP groups were evenly and densely distributed over the entire outer membrane of the schistosomula, including spines. The distance between the ferritin molecules and the parasite's surface was 24 +/- 5 nm, indicating that the lipid antigen had been incorporated into the outer membrane of the schistosomula.

Animals↗
PubMed1982

Protection against Schistosoma mansoni achieved by immunization with sonicated parasite.

Partial protection of (C57BL/6 X DBA/2)F1 mice against infection with Schistosoma mansoni has been achieved by immunization with small amounts (0.2 to 2.0 microgram) of crude cercarial sonicate adsorbed on aluminium hydroxide gel adjuvant (alum). A decrease of 34-90% in the adult worm burden, of the immunized mice, as compared to that of untreated mice or those injected with adjuvant alone, has been found in five experiments by liver perfusion six weeks after percutaneous challenge infection. High titers of anti-cercarial IgE antibodies have been found in the sera of the immunized mice by two independent techniques, radioimmunoassay and degranulation of rat basophilic leukemia cells (RBL-2H3), determined by 3H-serotonin release. By counting the live worms in the lungs of immunized and uniummized mice on days 4-7 after infection it was observed that the schistosomula were killed before they reached the lungs, probably at the skin. Mice immunized with the same amounts of cercarial antigen in Freund's complete adjuvant were not protected against infection with S. mansoni. These animals developed high titer of total anti-cercaria antibodies (determined by radioimmunoassay) but had low levels of antiparasite IgE. The possible role of IgE in protective immunity is discussed.

Aluminum Hydroxide↗
PubMed1980

Inactivation of the opiate receptor in bovine caudate nucleus by azide enkephalin analogs.

Photoaffinity labels have been prepared to react with the opiate receptor. The compounds are based on analogs of the enkephalins but involve significant changes in structure which allow introduction of azido substituents while still retaining binding affinity. The compounds designed have been shown to have biological activity in guinea pig ileum tests. A good correlation is obtained between the Kd for stereospecific binding and the Kd for activity in the guinea pig test. Affinity to and inactivation of the opiate receptor is obtained with two of the compounds which have affinities and activities comparable to morphine.

Affinity Labels↗
PubMed1980

A simple radioimmunoassay to determine binding of antibodies to lipid antigens.

A solid-phase radioimmunoassay to determine the binding of antibodies to lipid antigens is described. Polyvinyl chloride microtiter plates were coated with lipid antigens by placing in each well 25--100 microliters of solutions of the antigens in ethanol and evaporation of the solvent. The wells were than washed with 0.3% gelatine solution to remove loosely bound antigens and to saturate the plastic with an unrelevant protein to prevent unspecific binding of antibodies to the plates. About one-third of [3H] phosphatidylcholine adsorbed in this way (out of 1 nmole) was firmly attached to the plastic and was not washed away during a standard assay. The rest, loosely bound lipid, was washed away in the first 5 washings before the primary antibodies were applied. The technique is rapid and convenient and is as sensitive as a conventional solid-phase radioimmunoassay with protein antigens. It requires minute amounts of antigen. Less than 10(-12) moles of lipid antigen can be detected by this technique.

Animals↗
PubMed1978

Mechanism of action of papain: aryldehydroalanines as spectroscopic probes of acyl enzyme formation.

The alpha,beta-unsaturated aromatic amino acids phenyldehydroalanine (PDA) and styryldehydroalanine (SDA) were synthesized and used as sensitive spectroscopic probes to study the acylation of papain by specific substrates and inhibitors. The spectral changes observed upon acylation of the enzyme with peptides containing these amino acids are large red shifts of the absorption maxima (lambda max) of the chromophores. The magnitudes of the absorption shifts were 49 nm (from 277 to 326 nm) for PDA peptide and 59 nm (from 318 to 377 nm) for SDA peptides. The following specific substrates were synthesized: Ac-Phe-Phe-PDA-OEt, Ac-Phe-PDA-NH2, Ala-Ala-Phe-SDA-OME, Ala-Ala-Phe-SDA-NH2, Lys-Ala-(o-benzyl)tyrosyl-SDA-OMe, and Lys-Ala-(o-benzyl)-tyrosyl-SDA-NH2. Similarly, the specific competitive inhibitors Ac-Phe-PDA (Ki = 5.3 X 10(-6) M), Z-Phe-SDA (Ki = 5.6 X 10(-5) M), Ala-Ala-Phe-SDA (Ki = 2.9 X 10(-5) M), and Lys-Ala-(o-benzyl)tyrosyl-SDA (Ki = 1.1 X 10(-5) M) were prepared. An additional chromophore was used to follow the noncovalent association of an inhibitor or substrate with papain, independently from the acylation or deacylation steps. This chromophore, which was introduced into the peptides at position P2, IS p-(p"-dimethylaminophenylazo) phenylalanine (DAP). The light absorption spectrum of DAP is dependent on its environment. The substrates Ala-Ala-DAP-SDA-OMe and Ala-Ala-DAP-SDA-NH2 and the competitive inhibitor Ala-Ala-DAP-SDA (Ki = 2.5 X 10(-6) M) were prepared. The noncovalent binding of these peptides to the active site of papain was followed either by the increase in the absorption at 480 nm or the decrease at 550 nm. With these petides the acylation and deacylation reactions could be followed simultaneously at 377 nm. The extent of acyl enzyme formation was found to decrease in a sigmoidal way with increasing pH, with a transition point around pH 5.5.

Alanine↗
PubMed1977

A simple fluorescent method to determine complement-mediated liposome immune lysis.

A simple inexpective method is described to study the kinetics of complement-mediated immune lysis of liposomes containing sheep red blood cell lipid antigens. It is based on the fact that trapping the fluorescent molecule 1-aminonaphthalene-3,6,8-trisulfonate and the dynamic quencher, alpha, alpha'-dipyridinium p-xylene dibromide within the liposome inner volume results in an extinguished fluorescence signal. On addition of helmolysin plus active complement, liposome lysis occurs. The exit of the fluorophore and quencher and their subsequent dilution in the external volume abolishes the quenching, resulting in a high fluorescence signal. The details of the method are described as well as the initial kinetic results.

1-Naphthylamine↗
PubMed1977

Immunochemical determination of ganglioside GM2, by inhibition of complement-dependent liposome lysis.

Immunochemical quantitative determination of a lipid antigen, ganglioside GM2, has been developed, based on the inhibition of the immune lysis of liposomes containing the antigen in their lipid bilayer. It has been shown that the full expression of the antigenicity of the competing lipid requires its dispersion in accessory lipids. The assay of inhibition of liposome lysis can be used also for the establishment of the antigenic similarity of structurally related lipid antigens.

Animals↗