Harold W. Siegelman

Large-Scale Isolation of Intact Vacuoles and Isolation of Chloroplasts from Protoplasts of Mature Plant Tissues

Intact plant vacuoles were prepared in large numbers (10/sup 6/) from protoplasts of mature leaves, flower petals, stems, pedicels, filaments, styles, and young fruits. Treatment of protoplasts with 0.2 molar K/sub 2/HPO/sub 4/-HCl, pH 8, with slow stirring resulted in gentle osmotic rupture of the protoplasts and release of intact vacuoles. Particulate components of the protoplast, less the vacuole, were largely shed as an aggregate, which was removed by filtration. Vacuoles were recovered from the filtrate by low-speed centrifugation. The general procedure was also used to isolate chloroplasts with a high degree of integrity and excellent photochemical activity. (auth)

Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, 1298--1299). The ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue. 70--80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6mug/10(6) vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tuplipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for (Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K+, Na+, Mg2+, Cl-, and Ca2+ respectively, which are about the same as those in protoplasts.

The carotenoids of blue-green algae

Abstract The carotenoid compositions of Phormidium persicinum , P. luridum , P. faveolarum and Anabaena flos-aquae have been studied, both quantitatively and qualitatively. β-Carotene is the major carotenoid in all species. The xanthophylls comprise zeaxanthin, echinenone, canthaxanthin and the furanoid mutatochrome. Phormidium persicinum lacks glycosidic carotenoids. Myxoxanthophyll (myxol-2′-rhamnoside) and a 4-ketomyxol-2′-methylpentoside (tentatively 4-keto-myxoxanthophyll) are present in the other species. These distribution patterns are compared with those observed in other blue-green algae and some correlations with taxonomy are apparent.

Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts

Abstract Flavanone synthase, chalcone-flavanone isomerase and UDP-glucose; anthocyanidin-3- O -glucosyltransferase activities of protoplasts and subcellular fractions of protoplasts of Hippeastrum and Tulipa were investigated. Subcellular fractions studied were intact vacuoles, cytosol and particulate components of protoplasts less the vacuole. The cytosol fraction had the highest activity of the three enzymes studied. Results similar to those found for Hippeastrum were obtained with fractions from leaves and petals of Tulipa . The increase in flavanone synthase activity in the cytosol fraction from petals of Hippeastrum during development paralleled the increase in anthocyanin content of the petals.

The effects of single L-amino acid substitutions on the lethal potencies of the microcystins.

Microcystin-LR and -LA are more toxic than microcystin-LY and -RR in adult mice. They induce different degrees of thrombocytopenia and leukopenia, and the lethalities of their binary and ternary mixtures are addictive. Postnatal mice are resistant to doses of microcystin-LR that are lethal to adults but they are susceptible to higher doses. Substitution of a single L-amino acid for another in a microcystin markedly affects the dosimetric potency, but not the pathophysiology of its toxicity.

Lipopolysaccharides and polysaccharides of the cell envelope of toxic Microcystis aeruginosa strains

Lipopolysaccharides were obtained from three strains (PCC 7806, PCC 7820 and UV-017) of the waterbloom-forming cyanobacterium Microcystis aeruginosa. 3-Hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0) in addition to other fatty acids were identified in all three lipopolysaccharides. Glucosamine, the only amino sugar found, presumably represents the backbone amino sugar of the phosphate-free lipid A moiety. Heptoses were absent and 2-keto-3-deoxyoctonate was not detected in all three lipopolysaccharides. Strain-specificity was revealed in the complex composition of the polysaccharide moieties. Strains PCC 7806 and UV-017 were of the same chemotype, it differs from that of strain PCC 7820. Polysaccharides with strain-specific chemical compositions different from those of the respective lipopolysaccharides were obtained from each strain. The polysaccharides are likely to be ascribed to external cell envelope layers, their sugar specificity was in parallel with the O-chain chemotypes of the lipopolysaccharides of the three strains.

On the absolute configuration of 19′-hexanoyloxyfucoxanthin☆

Abstract The esterifying C 6 -acid in 19′-hexanoyloxyfucoxanthin has been identified as n -hexanoic acid by GLC of the methyl ester. Ozonolysis of 19′- n -hexanoyloxyfucoxanthin 3-benzoate provided the n -hexanoyloxy derivative of the allenic ketone produced from fucoxanthin 3-benzoate. NMR and CD correlation of the ozonolysis products and NMR of the native carotenoids provided the basis for assignment of the same absolute configuration of the 19′- n -hexanoyloxy derivative (3 S , 5 R , 6 S , 3′ S , 5′ R , 6′ S ) as for fucoxanthin. Biosynthetic implications are considered. CD data for 19′- n -hexanoyloxyfucoxanthin, fucoxanthin and some derivatives thereof are reported. Previously unreported minor carotenoids in Coccolithus huxleyi were diadinoxanthin and 3′-desacetyl 19′- n -hexanoyloxy-fucoxanthin.

Carotenoids of Anacystis nidulans, structures of caloxanthin and nostoxanthin

Abstract Reinvestigation of the carotenoids of Anacystis nidulans has confirmed the occurrence of β,β-carotene (β-carotene), β,β-caroten-3-ol (cryptoxanthin), β,β-carotene-3,3′-diol (zeaxanthin) and 2R,3R,3′R-β,β-carotene-2,3,3′-triol (absolute configuration assigned in the present work). In addition the previously unknown 2R,3R,2′R,3′R-β,β-carotene-2,3,2′,3′-tetrol has been isolated. The triol and the tetrol are considered identical with caloxanthin and nostoxanthin, respectively, for which allenic structures have been suggested by others. The chirality of these compounds followed from CD and 1 H NMR considerations.

Pathophysiology of cyanoginosin-LR: in vivo and in vitro studies

Cyanoginosin-LR, one of the group of virulent cyclic heptapeptide toxins (cyanoginosins) isolated from some strains of the cyanobacterium, Microcystis aeruginosa, kills mice within 1-2 hr after iv or ip injection. Although the liver is a target organ of the toxin, the rapidity of lethality is incompatible with metabolic death from failure of hepatocellular function. However, disintegration of sinusoidal endothelium causes massive intrahepatic hemorrhage. The loss of the structural integrity of hepatic sinusoids provides a previously undescribed mechanism for embolization of disintegrating cells from the liver to the lung. No injury to either cultured bovine pulmonary artery endothelial cells or mouse peritoneal macrophages was observed following prolonged incubation with high concentrations of the toxin, and there was no increase in vascular permeability to 125I-labeled albumin detected before intrahepatic hemorrhage. However, plasma fibronectin increased transiently after toxin injection. Acute, severe thrombocytopenia, a characteristic of cyanoginosin-LR toxicity, remains unexplained since platelets did not concentrate in the lungs, liver, or spleen. There are similarities between the effects of cyanoginosin-LR and of the lipopolysaccharide endotoxins, such as elevations of plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha.

Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa

Toxin-LR, a hexapeptide produced by Microcystis aeruginosa, causes marked hepatic vascular congestion, thrombocytopenia, microscopic pulmonary thrombi and death in 50-70 min when injected into mice. Although it is considered an hepatotoxin, we report that sublethal hepatocellular damage produced by CCl4 given 24 hr prior to toxin-LR administration prevents the acute deaths. However, CCl4-treated mice surviving toxin-LR acute effects often died during the subsequent three days. Pretreatment of mice with the microsomal enzyme inhibitors SKF 525A or cobaltous chloride did not alter the acute lethality of toxin-LR, but pharmacologic doses of hydrocortisone prevented both the acute and delayed deaths. X irradiation-induced thrombocytopenia or thrombocytopenia and leukopenia did not significantly affect the toxins lethality. In vitro platelet aggregation or lysis did not occur during incubation with toxin-LR, nor was a humoral aggregating factor detected in plasma from toxin-injected mice.