Glycosylphosphatidylinositol toxin of Plasmodium induces nitric oxide synthase expression in macrophages and vascular endothelial cells by a protein tyrosine kinase-dependent and protein kinase C-dependent signaling pathway.

In this study, we demonstrate that glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium falciparum origin responsible for nitric oxide (NO) production in host cells. Purified malarial GPI is sufficient to induce NO release in a time- and dose-dependent manner in macrophages and vascular endothelial cells, and regulates inducible NO synthase expression in macrophages. GPI-induced NO production was blocked by the NO synthase-specific inhibitor L-N-monomethylarginine. GPI also synergizes with IFN-gamma in regulating NO production. The structurally related molecules dipalmitoylphosphatidylinositol and iM4 glycoinositolphospholipid from Leishmania mexicana had no such activity, and the latter antagonized IFN-gamma-induced NO output. GPI activates macrophages by initiating an early onset tyrosine kinase-mediated signaling process, similar to that induced by total parasite extracts. The tyrosine kinase antagonists tyrphostin and genistein inhibited the release of NO by parasite extracts and by GPI, alone or in combination with IFN-gamma, demonstrating the involvement of one or more tyrosine kinases in the signaling cascade. GPI-induced NO release was also blocked by the protein kinase C inhibitor calphostin C, demonstrating a role for protein kinase C in GPI-mediated cell signaling, and by pyrrolidine dithiocarbamate, indicating the involvement of the NF-kappa B/c-rel family of transcription factors in cell activation. A neutralizing mAb to malarial GPI inhibited NO production induced by GPI and total malarial parasite extracts in human vascular endothelial cells and murine macrophages, indicating that GPI is a necessary agent of parasite origin in parasite-induced NO output. Thus, in contrast to dipalmitoylphosphatidylinositol and glycoinositolphospholipids of Leishmania, malarial GPI initiates a protein tyrosine kinase- and protein kinase C-mediated signal transduction pathway, regulating inducible NO synthase expression with the participation of NF-kappa B/c-rel, which leads to macrophage and vascular endothelial cell activation and downstream production of NO. These events may play a role in the etiology of severe malaria.

Proposed mechanism for the cytochrome P450-catalyzed conversion of aldehydes to hydrocarbons in the house fly, Musca domestica.

Experiments were performed to elucidate the mechanism of hydrocarbon formation in microsomal preparations from the house fly, Musca domestica. Antibody to both house fly cytochrome P450 reductase and a purified cytochrome P450 (CYP6A1) from the house fly inhibited (Z)-9-tricosene (Z9-23:Hy) formation from [15,16-3H]-(Z)-15-tetracosenal (24:1 aldehyde). Chemical ionization-gas chromatography-mass spectrometry (CI-GC-MS) analyses of the n-tricosane formed by microsomal preparations from [2,2-2H2,2-13C]- and [3,3-2H2,3-13C]tetracosanoyl-CoA demonstrated that the deuteriums on the 2,2- and 3,3-positions were retained in the conversion to the hydrocarbon product. Likewise, CI-GC-MS analysis of the Z9-23:Hy formed from [1-2H]tetracosenal by microsomal preparations demonstrated that the aldehydic proton on the 1-carbon was transferred to the hydrocarbon product. Hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were able to support hydrocarbon production from [3H]24:1 aldehyde in place of O2 and NADPH for short incubation times. From these data, a cytochrome P450 mechanism is proposed in which the perferryl iron-oxene, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the C=O double bond of the carbonyl group of the aldehyde. The reduced perferryl attacks the 1-carbon of the aldehyde to form a thiyl-iron-hemiacetal diradical. The latter intermediate can fragment to form an alkyl radical and a thiyl-iron-formyl radical. The alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2.

De novo biosynthesis of the aggregation pheromone components ipsenol and ipsdienol by the pine bark beetles Ips paraconfusus Lanier and Ips pini (Say) (Coleoptera: Scolytidae).

The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ips pini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1-14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified radiolabeled monoterpene alcohols from-males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.

Purification of a putative lipoprotein receptor from Schistosoma japonicum adult worms.

A 43-kDa putative lipoprotein receptor from Schistosoma japonicum adult worms (Sj43) has been purified by reverse-phase high-performance liquid chromatography (HPLC) using a Waters Delta-pak C4 300 A 15 mu 3.9 mm x 300 mm column. A linear acetonitrile gradient from 10-95%, spanning 40 min and at a flow rate of 0.9 ml min-1 was employed for the elution of bound material. Sj43 had a retention time of approximately 13 min on the column, whereas other main components from the parasite extract had a much longer retention time. Sj43 purified as a doublet which could be cleaved with Staphylococcus aureus V8 protease but was unaffected by treatment with a mixture of endoglycosidase F and glycopeptidase F. Human low-density lipoprotein exhibited typical saturation kinetics on the HPLC-purified Sj43 with a calculated stoichiometry of 2 mol LDL mol-1 of the putative receptor. No evidence of high-density lipoprotein (HDL3) saturation was observed on purified Sj43, this being of some interest since it parallels observations made with mammalian HDL3-binding proteins.

Contraindications in the use of the periodontal ligament injection.

How many times have practitioners used a periodontal ligament injection as a last resort or even in preference to the standard subcutaneous injection? In the past 5 years, the periodontal ligament injection has been implicated in compromising pulpal blood flow, damaging the developing crowns of underlying subcedaneous teeth, causing an increase in systemic absorption of epinephrine from the anesthetic solution, and causing resorption of the cementum. This article discusses some of the disadvantages concerning the periodontal ligament injection in view of some recent research findings.