Therapeutic uses of heparin beyond its traditional role as an anticoagulant.

A number of physiological effects have been ascribed to heparin since its discovery almost 80 years ago, many of which are independent from its first-described and best- characterized activity as an anticoagulant. Heparin and heparan sulphate are believed to possess many biological activities that include the ability to modulate embryonic development, neurite outgrowth, tissue homeostasis, wound healing, metastasis, cell differentation, cell proliferation and inflammation. In this review, David Tyrell, Stephen Kilfeather and Clive Page examine some of the activities of heparin (and heparin derivatives) beyond its effects as an anticoagulant, and discuss the therapeutic potential of this old, but certainly not antiquated, drug.

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

Relationship of the syntheses of spore coat protein and parasporal crystal protein in Bacillus thuringiensis.

Two major classes of polypeptides were extracted from the spore surface of Bacillus thuringiensis subsp. kurstaki: the 134,000-dalton protoxin that is the major component of the crystalline inclusion and spore coat polypeptides very similar to those found on Bacillus cereus spores. The quantity of spore coat polypeptides produced was reduced when compared with that produced by certain acrystalliferous mutants or by B. thuringiensis subsp. israelensis. The latter organism produced an inclusion toxic to mosquito larvae, but deposited very little of the inclusion protein on the spore surface. The reduction in spore coat protein in B. thuringiensis subsp. kurstaki was also seen in freeze-etched electron micrographs of spores. B. thuringiensis subsp. kurstaki spores germinated rather slowly when compared with related species, a property previously correlated with a deficiency or defect of the spore coat. Many mutants of B. thuringiensis subsp. kurstaki unable to form a crystalline inclusion were nontoxic and lacked a well-defined spore coat. Other mutants isolated either directly from the wild type or from coat-deficient mutants produced spores that were identical to those produced by the closely related species. Bacillus cereus, on the basis of morphology, germination rate, and the size and antigenicity of the spore coat polypeptides. Most of the protein extractable from the inclusion produced by B. thuringiensis subsp. israelensis was about 26,000 daltons, considerably smaller than the major polypeptide extractable from other inclusions. Some of the B. thuringiensis subsp. israelensis inclusion protein was found on the spore surface, but the majority of the extractable spore coat protein was the same size and antigenicity as that found on B. cereus spores. The B. thuringiensis subsp. israelensis spores germinated at a rate close to that of B. cereus, especially when the spores were formed at 37 degrees C, and the morphology of the spore surface was very similar to that of B. cereus.

Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains.

Parasporal crystals of Bacillus thuringiensis subspp. kurstaki, tolworthi, alesti, berliner, and israelensis were compared by electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis, tryptic peptide mapping, immunological analysis, and insecticidal activity. Spore coats also were compared by polyacrylamide gel electrophoresis. B. thuringiensis subsp. israelensis crystals were lethally toxic to mosquito larvae and nontoxic to tobacco hornworm larvae. Conversely, crystals from the other subspecies killed tobacco hornworm larvae but were ineffective against mosquitoes. Crystalline inclusion bodies of all subspecies contained a protoxic subunit that had an apparent molecular weight of approximately 1.34 X 10(5). However, polyacrylamide gel electrophoretic patterns of solubilized crystals revealed a small-molecular-weight component (apparent molecular weight, 26,000) in B. thuringiensis subsp. israelensis that was absent in the other subspecies. Also, differences were noted in amino acid composition and tryptic peptide fingerprints. Crystal proteins were found in spore coats of all subspecies. The results suggest that insecticidal specificity is due to unique polypeptide toxins.

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.