Dolichyl-phosphomannose synthase from the Archae Thermoplasma acidophilum

Archae (formerly Archaebacteria) comprise an entire kingdom of organisms placed halfway between prokaryotes and eucaryotes in evolution, This class of organisms lacks murein cell wall and is devoid of organelles, yet Archae synthesize and export N-linked and O-linked glycoproteins utilizing only the plasma membrane. Study of glycosylation systems in Archae is extremely interesting because the plasma membrane must perform many functions normally carried out by the endoplasmic reticulum and Golgi in eucaryotes. This report represents the first glycosyl transferase system enzyme demonstrated from archae showing a functional relationship with homologous eucaryotic enzymes. Archae dolichyl-phosphoryl-mannose synthase was purified 1070-fold from Thermoplasma acidophilum by column chromatography on Sephacryl S-200, Cibacron blue 3GA-agarose, Octyl-Sepharose, and hydroxylapatite in the presence of 0.2% polioxyethylene 9 lauryl ether. The enzyme activity was stimulated by MgCl, (20 mM optimum) and exhibited a pH optimum at 6.0. Although the native polyisoprenol has not been isolated or characterized, the enzyme prefers dolichyl phosphate (dol-P) to C55-polyisoprenol as an acceptor, and the K(m) value for dol-P was calculated to be 2.6 μM. Amphomycin, an inhibitor of dol-P-Man synthase, blocked mannosyl transfer to the endogenous lipids, proteins, and to dol-P; 100 μg/ml amphomycin inhibited 97% of mannosyl transfer to dol-P, and 50% to endogenous accepters, indicating direct transfer from GDP-mannose to some intermediates or final structures. The size range of [3H]Man-oligosaccharides from acid-labile manno-lipid product was from dp 1 to 4. dol-P-Man synthase activity could be correlated directly with a 42 kDa band on SDS/polyacrylamide gel electrophoresis

Intrinsic tryptophan fluorescence measurements suggest that polylactosaminyl glycosylation affects the protein conformation of the gelatin‐binding domain from human placental fibronectin

Glycosylation can affect the physical and biochemical properties of the polypeptide chain in glycoproteins. Asparagine‐N‐linked polylactosaminyl glycosylation of the chymotryptic 44‐kDa gelatin‐binding domain from human placental fibronectin confers protease resistance [Zhu, B. C. R., Fisher, S. F., Panda, H., Calaycay, J., Shively, J. E. & Laine, R. A. (1984) J. Biol. Chem. 259, 3962–3970] and weakens the binding to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041–4045]. Intrinsic tryptophan fluorescence of the gelatinbinding domain was used to probe glycosylation‐dependent protein conformation changes. In gelatin‐binding fragments containing incrementally smaller polylactosamine oligosaccharides, the fluorescence intensity progressively decreased and the emission spectrum shifted about 7 nm to the blue. Removal of the polylactosamine chains from a highly glycosylated fragment with endo‐β‐galactosidase from Escherichia freundii also quenched the protein fluorescence. The fluorescence lifetimes did not appear to be affected by the extent of glycosylation, suggesting static quenching of the tryptophan emission in the low glycosylated fragments. Acrylamide quenching studies showed that the accessibility of the tryptophans to small solutes was not altered by glycosylation. The steadystate emission anisotropy increased with decreasing polylactosamine chain length. The results indicate that the polylactosamine chains alter the tryptophan environments in the gelatin‐binding domain, probably by changing the polypeptide conformation. These putative protein conformation changes may be partially responsible for the altered gelatin binding, protease resistance, and cell adhesion functions of fetal tissue fibronectin. Copyright © 1990, Wiley Blackwell. All rights reserve

Toxicity and behavioral effects of nootkatone, 1,10-dihydronootkatone, and tetrahydronootkatone to the formosan subterranean termite (Isoptera: Rhinotermitidae)

Toxicity and behavioral effects of nootkatone and two of its derivatives, 1,10-dihydronootkatone and tetrahydronootkatone, to Coptotermes formosanus Shiraki were investigated on workers from two different colonies by using topical application assays, repellency assays, and sand barrier assays. The acute toxicity of the nootkatones on workers from both colonies increased as the saturation of the molecule increased, but the difference was significant for only one colony. The results of the repellency assays showed a similar trend of efficiency; the threshold concentration for significant repellency was four-fold higher in nootkatone treatments (50 ppm) than in the reduced derivatives 1,10-dihydronootkatone or tetrahydronootkatone (12.5 ppm). In sand barrier assays, a concentration of 100 ppm of any of the three chemicals significantly reduced termite survival, tunnel building, and food consumption after a 12-d exposure. Termites preexposed tolOO ppm nootkatone-treated sand and placed in containers without nootkatone for 15 d continued to exhibit abnormal feeding and digging behaviors; survivorship, tunneling, and feeding activities were significantly reduced by 83.5, 63.2, and 95.4%, respectively. Termites pretreated for 12 d at concentrations of 50 and 75 ppm nootkatone and tetrahydronootkatone returned to normal digging activity after they were removed from the treatments, but their feeding activity was significantly reduced

Evaluation of Vetiver Grass Root Growth, Oil Distribution, and Repellency Against Formosan Subterranean Termites

The growth rate of vetiver grass [Vetiveria zizanioides (L.) Nash (Graminales: Poaceae)], roots, and oil distribution were evaluated in an 8-month field study. The amount of vetiver oil present in the root system increased with each sampling date. In December, the final sampling period, mean root weight increased 520% from the previous sampling period (October). At the end of the study, root growth measured over 2 m long and 25 cm wide and weighed 0.48 kg (dry weight). In addition, a laboratory study was conducted to determine if the roots of vetiver grass when used as mulch, are effective against Formosan subterranean termites, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). In past studies, chemical components in the roots of this grass were shown to be effective repellents and toxicants to Formosan subterranean termites. In the present study, the 25% vetiver root mulch treatment proved to decrease tunneling activity and wood consumption and increase termite mortality. These results provide preliminary evidence that vetiver roots may have use as an additive to garden mulches against termites

Terpene-lnduced Morphological Changes to Exoskeleton of Formosan Subterranean Termites (Isoptera: Rhinotermitidae): Toxic Effects of cis-nerol

The terpenoid, cis-nerol was found to be highly toxic to the Formosan subterranean termite, Coptotermes formosanus Shiraki. Morphological abnormalities were observed in the exoskeleton including the spiracles and trichoid sensilla using scanning electron microscopy after exposure of termites to cis-nerol for 2 h. Proteins were found in a water-soluble fraction collected from the whole termite body exposed to cis-nerol for 60 min. It appears that terpenoids cause damage to cell membranes resulting in a significant loss of proteins