Faculty Bibliography

In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis. We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated. The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ('ARG boxes'). The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined. Antibodies prepared against the fusion protein react with the repressor. The repressor is precipitable by L-arginine, which facilitates its purification. The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500. To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR

A cytolytic toxin from the basidiocarps of the edible mushroom Flammulina velutipes was purified to homogeneity. The lysin, flammutoxin, is a single polypeptide chain of Mr 32,000 and pK about 5.4. It contains unusually large amounts of tryptophan, serine and glycine, and few or none of the sulfur-containing amino acids. Erythrocytes of rat, rabbit, guinea pig, man, mouse, cat and dog were sensitive to lysis, in that order, whereas erythrocytes of sheep, ox, goat, swine and horse were largely or completely resistant to lysis. The toxin appears not to be a phospholipase and it was not inhibitable by any of a variety of lipids. Hemolysis probably involves alteration of the erythrocyte membrane, with formation of submicroscopic ion channels, and it appears to be of the osmotic type. In some respects flammutoxin resembles phallolysin, a cytolytic toxin obtained from the mushroom Amanita phalloides

There has been controversy whether the plasma protein, alpha 1-acid glycoprotein (AGP), is able to inhibit invasion of erythrocytes by P. falciparum merozoites. Because AGP resembles a typical cell membrane sialoglycoprotein, it has been proposed that it can inhibit the parasite from interacting with its sialoglycoprotein receptor on the erythrocyte surface. We therefore isolated and tested samples of AGP obtained from a series of separate individuals. For comparative purposes, we also tested AGP prepared from the plasma of patients with elevated levels of AGP, as well as AGP obtained from two commercial sources. The authenticity and purity of the AGP samples was established by SDS-PAGE, radial immunodiffusion, and crossed immunoelectrophoresis. Our results indicated that none of the nine samples tested had any significant inhibitory effects in our P. falciparum invasion assay system

Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay

Solubilized preparations of purified glycophorins and specific domains of these molecules were assessed for their effects as inhibitors of Plasmodium falciparum invasion of human erythrocytes in vitro. The ability of newly invaded merozoites to continue developing and incorporating [3H]hypoxanthine during a 24-h period after their invasion was used as an assay for merozoite invasion. Glycophorins A, B, and C were found to be equally effective as inhibitors. Previous studies had shown N-acetylglucosamine, a sugar component of glycophorins A and C but not B, to be an effective inhibitor. Accordingly, molecular domains common to all of the glycophorins were further assessed. Sialic acid was shown to act almost as effectively as N-acetylglucosamine, presumably because of the structural similarities between these sugars. The inhibitory ability of sialic acid is considerably enhanced when presented to the parasite in a clustered form, as in an oligosaccharide. The acetyl group of these sugars does not appear to play an essential role in this inhibition. How the P. falciparum merozoite recognizes and interacts with the sugar domains of the glycophorin molecule remains to be determined

A method is described for the rapid purification of serologically active high titer anti-I and anti-i cold antibodies from the sera of patients with chronic cold agglutinin disease (CCAD). The purification procedure is based on thermal affinity chromatography, using desialated orosomucoid (alpha 1-acid glycoprotein)-Sepharose 4B conjugated beads. The nature of the interaction between the cold agglutinins (CA) and the desialated orosomucoid is unknown. Inhibition studies, however, revealed that the cold hemagglutinating activities of all the anti-i sera were inhibited by desialated orosomucoid while only 1 out of 4 of the anti-I sera was similarly affected. Anti-I or anti-i antibodies were separated from whole sera in 7 out of 7 samples with a recovery in most cases of 100% of the cold hemagglutinating activity. The resultant products were purified monoclonal IgM fractions which could react with anti-kappa and anti-mu but not with anti-lambda sera. The homogeneity, purity and specificity of all preparations were confirmed by immunodiffusion analysis against purified I and i blood group antigens isolated from human erythrocyte membranes, zonal and right-angle electrophoresis and hemagglutination or hemagglutination inhibition studies.

Three red blood cell membrane fractions (Band 3, macromolecules exhibiting I antigenic determinants, and a delipidated glycoprotein fraction) were separated from red blood cell membranes and tested for their ability to inhibit penetration of red blood cells by Plasmodium falciparum merozoites in an in vitro inhibition assay. The delipidated glycoprotein fraction (containing the major sialoglycoproteins and devoid of Band 3) was the only fraction that inhibited merozoite invasion. This fraction showed 73% and 70% inhibition at 1 mg/ml and 500 micrograms/ml, respectively, and slight inhibition below these levels