Martin S. Nachbar

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.

Purification of a hemagglutinin from Limulus polyphemus by affinity chromatography.

Summary A. hemagglutinin has been isolated from the hemolymph of the common horseshoe crab ( Limulus polyphemus ) using affinity chromatography as the primary purification procedure. The hemagglutinin specifically binds to a bovine submaxillary mucin (BSM) affinity gel and is subsequently released upon the addition of Na citrate to the elution buffer. The purity of the final product was attested to by polyacrylamide gel electrophoresis, electron microscopic observation and by a greater than 1500-fold increase in specific activity as compared to the starting material. The purified hemagglutinin was able to give a precipitin reaction in immunodiffusion gels against purified BSM but not against desialated BSM.

Can a web-based curriculum improve students' knowledge of, and attitudes about, the interpreted medical interview?

AbstractOBJECTIVES: To develop and evaluate a web-based curriculum to introduce first year medical students to the knowledge and attitudes necessary for working with limited English proficient (LEP) patients through interpreters. METHOD: Six hundred and forty first year medical students over 4 consecutive years took this curriculum as part of their Patient Physician and Society course. They viewed 6 patient-physician-interpreter video vignettes, gave open text analyses of each vignette, and compared their responses to those generated by experts, thereby receiving immediate formative feedback. They listened to video commentaries by a cultural expert, lawyer, and ethicist about working with LEP patients, completed pre- and postmodule questionnaires, which tested relevant knowledge and attitudes, and were provided a summative assessment at the end of the module. Students completed an optional survey assessing the educational value of, and providing open text commentary about, the module. RESULTS: Seventy-one percent (n=456) of first year students who completed the module consented to have their data included in this evaluation. Mean knowledge (19 items) scores improved (46% pre- to 62% postmodule, P<.001), reflecting improvements in knowledge about best interpreter practices and immigration demographics and legal issues. Mean scores on 4 of 5 attitude items improved, reflecting attitudes more consistent with culturally sensitive care of LEP patients. Mean satisfaction with the educational value of the module for 155 students who completed the postmodule survey was 2.9 on a scale of 1 to 4. CONCLUSION: Our web-curriculum resulted in short-term improvement in the knowledge and attitudes necessary to interact with LEP patients and interpreters. The interactive format allowed students to receive immediate formative feedback and be cognizant of the challenges and effective strategies in language discordant medical encounters. This is important because studies suggest that the use of these skills in patient encounters leads to greater patient and provider satisfaction and improved health outcomes.

The use of lectins in the quantitation and analysis of macromolecules by affinoelectrophoresis.

The technique of rocket affinoelectrophoresis, initially introduced for the quantitation of a succinylated mannan by concanavalin A [Owen, P., and Salton, M. R. J. (1976) Anal. Biochem.73, 20–26] has been extended to (a) the quantitation of four other macromolecules: vz. streptococcal lipoteichoic acid, I blood group substance, desialylated bovine submaxillary mucin, and desialylated pig submaxillary mucin; and (b) the use of three other lectins: vz. wheat germ agglutinin, soybean agglutinin and peanut agglutinin. In all cases stable-affinity precipitin rockets were observed the heights of which bore an approximately linear relationships to the amount of sample analyzed. For all lectins, the detection limits were in the range of 15–25 ng. Furthermore, a new technique has been introduced called crossed affinoelectrophoresis which is basically a two-dimensional variant of rocket affinoelectrophoresis. This technique can be used with concanavalin A, wheat germ agglutinin, soybean agglutinin, or peanut agglutinin in the affinity gel and allows the examination of glycoprotein homogeneity. Modification of this technique, involving the use of other lectins or antiserum in intermediate gels, is also described and evaluated.

Characteristics of a lipid-rich NADH dehydrogenase-containing particulate fraction obtained from Micrococcus lysodeikticus membranes

Abstract A fraction of homogenous particle size, enriched in lipid and NADH dehydrogenase has been obtained from Micrococcus lysodeikticus membrane by treatment with EDTA. Lipid activation of NADH dehydrogenase activity of a n -butanolextracted EDTA wash has been demonstrated. The relationship of this fraction to other fractions obtained by mild procedures from this organism is discussed and a possible structure for the membrane is proposed.

The production and purification of specific anti-soybean agglutinin antibody by affinity chromatography.

Abstract Modifications of previously reported procedures to purify soybean agglutinin were used to prepare a protein from soybean meal which agglutinated Ehrlich ascites carcinoma cells. This agglutination was inhibited by concentration of N- acetyl- D -galactosamine as low as 5 · 10−7 M. The purity of the final product was assussessed by polyacrylamide gel eletrophoresis in both sodium dodecyl sulfate and Trisglycine systems. The purified soybean agglutinin was strongly immunogenic in rabbits, yielding high titer antisera. Specific rabbit anti-soybean agglutinin γ-globulin was isolated utilizing a soybean agglutinin Sepharose affinity column. This interaction of soybean agglutinin and antibody was not inhibited by N- acetyl- D -galactosamine .

The effect of aliphatic alcohols upon the dissociation of Micrococcus lysodeikticus membrane lipids and proteins.

Abstract The ability of aliphatic alcohols to extract 32 P-labeled phospholipid from aqueous dispersions of Micrococcus lysodeikticus membranes into the organic solvent phase has been investigated and the following order of efficiency of extraction observed: tert -amyl alcohol = n -butanol = iso -butanol = sec -butanol > n -amyl alcohol > iso -amyl alcohol > 3-pentanol. This order approximates that of the water solubility of these alcohols. Inclusion of urea (2 M or 6 M) or NaCl (0.1 M or 1.0 M) in the aqueous phase reduced the extractibility of 32 P-labeled lipid into the organic solvent phase. Residual 32 P-labeled material in the aqueous phase following n -butanol extraction could in part be accounted for as phospholipid. Almost maximal recovery of the phospholipid in the solvent phases occurred when aqueous dispersions of membrane lipid and bovine serum albumin (protein: lipid ratio, 2:1) were extracted. The recovery of protein in the aqueous phase was about 45–50 % for all solvents and thus represents a much lower value for “soluble” protein than that observed with erythrocyte membranes. The lower recovery of soluble proteins in this bacterial membrane system is probably due to the presence of insoluble aggregates of the electron transport components of the membrane. Addition of 2 M urea increased solubilization of protein, whereas 6 M urea and 0.1 and 1.0 M NaCl decreased the aqueous phase protein. ATPase activity survived extraction with a number of the alcohols, being less sensitive to those with the hydroxyl group on carbon-1; it was largely inactivated by urea and NaCl additions to the aqueous phase. NADH-dehydrogenase showed good preservation following extraction with tert -amyl alcohol in the presence of 2 M urea.

Educational imperatives drive technological advancement in the surgery clerkship.

quiz mode students are asked to correctly identify structures drawn at random for that panel. Up to 6 multiple-choice question flags are included for each image, with random sequencing of options on repeated attempts. The program also generates an index allowing students to quickly locate structures of interest anywhere in the program. Academic staff can produce each image panel within 30–50 minutes, depending on the number of structures and level of question difficulty. Evaluation of results Approximately 250 copies of the program are produced and distributed on demand each year, with students sharing copies between colleagues. Ready availability of the program reduces wear and tear on precious human prosections, while providing students with a convenient revision tool for use at home. The generic nature of the template means that units can be easily tailored for specific groups of students using actual teaching material they will encounter during assessment.

Identification and Quantitation of Solubilized I blood Group Substance by Wheat Germ Agglutinin Using Quantitative Immunoelectrophoresis

Wheat germ agglutinin (WGA) has been shown to react specifically with solubilized I blood group substance, purified from papain treated human erythrocyte membranes. WGA and I react to form an affinity precipitate in immunodiffusion gels, a reaction which can be blocked by the incorporation of N-acetyl glucosamine into the gel. The I material was a strong inhibitor of both anti-I cold hemagglutination and WGA hemagglutination reactions. Utilizing the techniques of crossed immunoelectrophoresis we have clearly established that WGA and anti-I IgM cold antibody are reacting with the same membrane macromolecule (I antigen). WGA was then used in a rocket affinoelectrophoretic assay system to quantitate I substance. The limits of detection in this system was 25 ng.

Immunochemistry and peptide mapping of Micrococcus lysodeikticus membrane proteins

Abstract Three proteins, ATPase (EC 3.6.1.3), NADH dehydrogenase (EC 1.6.99.3) and a component migrating rapidly on electrophoresis in polyacrylamide gel (‘fast-moving component’) have been purified from isolated membranes of Micrococcus lysodeikticus . Immunochemical analysis of these proteins demonstrated that each protein possessed a unique antigenic specificity when tested against antiserum to M. lysodeikticus membranes. Treatment of the proteins with sodium dodecyl sulfate revealed a common antigenic specificity. The common antigen was not detectable in catalase purified from the cytoplasmic fraction of M. lysodeikticus and is considered to be peculiar to the membrane proteins. It is heat labile and destroyed by trypsin or by pronase. Trypsin digestion of the purified proteins yielded peptide “fingerprints” showing the presence of common major peptides. Some minor peptides appeared to be unique for each protein. Purified sodium dodecyl sulfate subunit of ATPase and the fast-moving component had strikingly similar peptide fingerprints, although their migration in polyacrylamide gels differed. Common peptides were also obtained from tryptic digests of whole membranes and the deoxycholate-insoluble residue containing electron transport components. In contrast to the membrane fractions the purified cytoplasmic protein catalase, gave an entirely different pattern of peptides on tryptic digestion and fingerprinting.