Faculty Bibliography

IgD receptors on murine T cells have been reported in this issue [Tamma, S. M. L., Amin, A. R., Finkelman, F. D., Chen, Y.-W., Thorbecke, G. J. & Coico, R. F. (1991) Proc. Natl. Acad. Sci. USA 88, 9233-9237] to bind either the first or third constant region of the heavy-chain of IgD molecules--findings that could not be satisfactorily explained by IgD amino acid sequences. We now find that boiled IgD molecules or low-Mr fragments from protease-digested IgD still inhibit binding of IgD-coated erythrocytes to IgD receptors. This inhibitory activity can be absorbed with the murine IgD-binding lectin from Griffonia simplicifolia 1 (GS-1) immobilized on Sepharose. N-linked glycans, obtained from N-glycanase-treated IgD and purified by binding to GS-1-Sepharose, also inhibit rosette formation of T-helper cells bearing receptors for IgD with IgD- or mutant IgD-coated erythrocytes. Deglycosylated IgD, produced by treatment with N-glycanase, no longer binds to the lectin and fails to inhibit IgD rosetting. Binding of intact IgD to T cells is also competitively inhibited by N-acetylgalactosamine, galactose, N-acetylglucosamine, and neoglycoproteins containing these sugars. These results clearly show that N-linked glycans, present in both the first and third constant regions of the delta heavy-chain domains, are prerequisites for binding of IgD to IgD receptors

The initiator protein RepA1 of the IncFII replicon RepFIC derived from the enterotoxin plasmid EntP307 has been cloned under the control of the lambda PL promoter. This has enabled us to overproduce this protein and study its properties. Here we show that RepA1 is a soluble basic protein with an experimentally determined molecular weight of 40,000. Deletion analysis indicates that the overproduced protein originates from the open reading frame which we previously designated as coding for RepA1. We have also shown that the replication function of the replicon RepFIC depends on the intact RepA1 coding frame

The sin gene of Bacillus subtilis encodes a dual-function regulatory protein, Sin, which is a negative as well as a positive regulator of alternate developmental processes that are induced at the end of vegetative growth in response to nutrient depletion. Sin has been purified to homogeneity by using a simple two-step procedure. It was found to bind to the developmentally regulated aprE (alkaline protease) gene at two sites in vitro. The stronger Sin-binding site (SBS-1) is located more than 200 bp upstream from the transcription start site. It is required for Sin repression of aprE expression in vivo, as strains bearing SBS-1 deletions were not affected by the sin gene. The second, weaker Sin-binding site lies on a DNA fragment that contains the aprE promoter. Results of DNase I, exonuclease III, and dimethyl sulfate footprinting analysis of SBS-1 suggested that Sin binding involves two adjacent binding sites which appear to contain two different partial dyad symmetries. An analysis of the predicted amino acid sequence of Sin revealed a potential leucine zipper protein dimerization motif which is flanked by two helix-turn-helix motifs that could be involved in recognizing two different dyad symmetries

Hemozoin, the pigment granule which develops within the blood stage food vacuole of the malaria parasite Plasmodium falciparum, was biochemically characterized. Hemozoin was found to be composed of 65% protein, 16% ferriprotoporphyrin-IX (hematin), 6% carbohydrate, and trace amounts of lipid and nucleic acids. The overwhelming majority of the protein component is a mixture of native and denatured human globin non-covalently associated with the metalloporphyrin. Immunoelectron microscopy, employing anti-human hemoglobin as a probe, identified in situ association of hemoglobin with hemozoin. Hemozoin produced within diabetic blood had a higher proportion of carbohydrate, suggesting that the carbohydrate component comes from non-enzymatic glycosylation of hemoglobin

The alpha-D-galactopyranosyl binding lectin from the seeds of Bandeiraea simplicifolia (a.k.a. Griffonia simplicifolia) termed BS-I, strongly reacts with murine IgD and with no other protein in ascites including all other classes of immunoglobulins as determined by immunoprecipitation, hemagglutination inhibition and affinity binding. Based on this finding, murine IgD could be rapidly purified directly from whole ascitic fluid by passage over affinity beads of BS-I linked to Sepharose 4B and subsequent elution by a buffer containing 0.1 M D-galactose. The sugar eluted product is 95-99% pure as determined by SDS-PAGE and represents 90-95% of the total IgD in the initial ascites by ELISA assay. Both monomeric and dimeric murine IgD may be purified by this procedure. Human IgD is unreactive with this lectin. Treatment of purified IgD with endoglycosidases that remove either O- or N-linked glycosides indicates that BS-I binds to IgD only via N-linked carbohydrate chains

Two different lectins (termed BnA-I and BnA-II) with distinct carbohydrate specificities were identified and subsequently isolated from the marine bryozoan Bugula neritina. BnA-I hemagglutinating activity was inhibited by N-acetylated hexosamines, their polymers, and glycoproteins rich in these moieties. BnA-II-induced hemagglutination was not blocked by any simple sugars but could be inhibited by several complex glycoproteins (e.g., thyroglobulin and orosomucoid). Both lectins required the presence of Ca(+)+ for reactivity and were purified by affinity chromatographic procedures. Purified BnA-I was determined to have a native molecular weight of 240 Kd and appeared to be a hexameric homopolymer while BnA-II was shown to be a 65-70 Kd monomer. Both lectins showed seasonality in expression, BnA-I appearing in animal extracts prepared in the spring and fall while BnA-II was expressed only during the summer and winter

This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the BglII site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong pL promoter. Northern (RNA) blotting assays revealed a 10- to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-beta-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-beta-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to keyhole limpet hemocyanin

Significant amounts of an estradiol-binding protein (EBP) are present in pancreatic acinar cells. This protein differs from the one found in female reproductive tissues and secondary sex organs (which is commonly referred to as estrogen receptor). EBP has now been purified from rat pancreas and was used as an antigen to induce polyclonal antibodies in rabbits. The antiserum obtained was purified initially by ammonium sulfate fractionation and then still further by interaction with a protein fraction from pancreas that was devoid of estradiol-binding activity. The latter procedure was used to precipitate nonspecific immunoglobulin Gs. Western blot analysis demonstrated that the anti-EBP antibody reacted specifically with a doublet of protein bands having mol wt of 64K and 66K. When this purified antibody was used as an immunocytochemical probe in conjunction with protein-A-gold, acinar cells were labeled on the surface of the endoplasmic reticulum, on the plasma membrane, and in mitochondria. This specific labeling pattern was not observed when preimmune serum was used. No labeling was observed over the nucleus, Golgi apparatus, or zymogen granules with purified anti-EBP antibodies. The unexpected distribution of EBP in both the endoplasmic reticulum and mitochondria is discussed

The kinetics of hemolysis resulting from the action on rabbit erythrocytes of a highly purified cytolytic toxin (26,000 mol. wt) isolated from a spore-crystal mixture of Bacillus thuringiensis israelensis was studied. Course of hemolysis, as determined by release of hemoglobin, yielded sigmoid curves whose maximum slopes were taken as a measure of the rate of lysis. Hemolysis occurred without an induction period, and the rate of lysis was a linear function of toxin concentration. Rate of hemolysis as a function of temperature yielded an Arrhenius constant of 9300 calories per mole. The toxin was active between pH 4.5 and 8.0. Lysis was strongly inhibited by Cu2+, Fe2+ and Zn2+ in concentrations as low as 0.025 M. Phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin inhibited lysis, whereas phosphatidylethanolamine, cerebroside, cholesterol and major integral erythrocyte membrane proteins caused little or no inhibition. Inhibition of lysis by sucrose indicates that hemolysis is of the colloid-osmotic type

A hemolytic toxin from Bacillus thuringiensis israelensis was obtained by alkaline extraction and fast protein liquid chromatography (chromatofocusing followed by gel filtration). The toxin displayed a pI of 4.6-4.8 and an Mr of 26,000. Amino acid analysis demonstrated large amounts of serine, glycine and glutamic acid. The toxin was strongly lytic for rabbit, human and non-human primate erythrocytes, and was weakly lytic toward equine, feline, canine, bovine, amphibian and reptilian erythrocytes. It was weakly entomocidal as indicated by a lethal potency (LC50) of 25 micrograms/ml for second instar larvae of Anopheles stephansi. Direct micro-ELISA indicated that the toxin lacked antigenic identity with numerous eukaryotic and prokaryotic toxins and venoms