Ganglioside/protein kinase signals triggering cytoskeletal actin reorganization

Exposure of neuronal cells to nanomolar concentrations of oligosaccharide portions of ganglioside GM2 and GT1b stimulates cAMP-dependent protein kinase (PKA) Ca2+/calmodulin-dependent protein kinase II (CaMKII), respectively, in a few seconds suggesting the presence of glyco-receptor-like molecules on the surface of the cells. Both GM2/PKA (GalNAc/PKA) and GT1b/CaMKII signaling cascades induced cytoskeletal actin reorganization through Cdc42 activation leading to filopodia formation within 2 min. Long-term effects of these glyco-signals were facilitation of dendritic differentiation of primary cultured hippocampal neurons and cerebellar Purkinje neurons indicating physiological roles of the signals in neuronal differentiation and maturation.     

An electrophoretic profiling method for thiol-rich phytochelatins and metallothioneins

Thiol-rich peptides such as phytochelatins (PCs) and metallothioneins (MTs) are important cellular chelating agents which function in metal detoxification and/or homeostasis. The variations in molecular sizes and lack of chromophores of these peptides make their analysis difficult. This paper reports an electrophoresis-based method for a broad screen of thiol-rich peptides and proteins. The method uses the thiol-selective fluorescent tag, monobromobimane, coupled with Tricine--sodium dodecyl sulphate--urea polyacrylamide gel electrophoresis for a sensitive determination of both PCs and MTs. Results for PCs were confirmed by two-dimensional NMR and HPLC-tandem MS analyses. Sample throughput is substantially improved over chromatography-based methods through parallel sample analysis in 1 h of electrophoretic separation. The method is versatile in that peptides ranging from glutathione to large proteins can be analysed by simple modification(s) of the extraction and electrophoretic conditions, and the nature of the method supports serendipitous detection of unexpected or novel thiol metabolites.     

Liquid chromatographic determination of 1-adamantanamine and 2-adamantanamine in human plasma after pre-column derivatization with o-phthalaldehyde and 1-thio-beta-D-glucose

We investigated high-performance liquid chromatographic (HPLC) determination of 1-adamantanamine hydrochloride (1-ADA) and 2-adamantanamine hydrochloride (2-ADA) in human plasma after the derivatization with o-phthalaldehyde (OPA) and 1-thio-beta-D-glucose (TG). Extracted human plasma samples were mixed with OPA and TG at room temperature for 6 min and injected onto HPLC. Retention times of 1-ADA and 2-ADA derivatives were 12.6 and 14.1 min, respectively. The lower limits of detection of 1-ADA and 2-ADA were 0.02 and 0.008 microg/ml, and the lower limits of quantitation of 1-ADA and 2-ADA were 0.025 and 0.01 microg/ml, respectively. The coefficients of variation for intra-day and inter-day assay of 1-ADA and 2-ADA were less than 4.4 and 6.0%, respectively. L-Dopa and dopamine were not found to interfere with the peaks of 1-ADA and 2-ADA derivatives. Human plasma unbound fraction (f(p)) values of 1-ADA varied between 0.32 and 0.48, while those of 2-ADA varied between 0.38 and 0.68. These results indicate that HPLC assay of 1-ADA and 2-ADA by derivatization with OPA and TG is simple, rapid, sensitive and reproducible for determining 1-ADA and 2-ADA in human plasma.     

Secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza A virus infection in the airway and chemical agents up-regulating their levels may have therapeutic potential

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Entry of this virus into cells is primarily determined by host cellular trypsin-type processing proteases, which proteolytically activate viral membrane fusion glycoprotein precursors. Human IAV and murine parainfluenza virus type 1 Sendai virus are exclusively pneumotropic, and the infectious organ tropism of these viruses is determined by the susceptibility of the viral envelope glycoprotein to cleavage by proteases in the airway. Proteases in the upper respiratory tract are suppressed by secretory leukoprotease inhibitor, and those in the lower respiratory tract are suppressed by pulmonary surfactant, which by adsorption inhibits the interaction between the proteases and viral membrane proteins. Although the protease activities are predominant over the activities of inhibitory compounds under normal airway conditions, intranasal administration of inhibitors was able to significantly suppress multi-cycles of viral replication in the airway. In addition, we identified chemical agents that could act as defensive factors by up-regulating the levels of the natural inhibitors and immunoglobulin A (IgA) in airway fluids. One of these compounds, ambroxol, is a mucolytic and anti-oxidant agent that stimulates the release of secretory leukoprotease inhibitor and pulmonary surfactant in the early phase, and IgA in the late phase of infection at an optimal dose, i.e. a dose sufficient to inhibit virus proliferation and increase the survival rate of animals after treatment with a lethal dose of IAV. Another agent, clarithromycin, is a macrolide antibiotic that increases IgA levels through augmentation of interleukin-12 levels and mucosal immunization in the airway. In addition to the sialidase inhibitors, which prevent the release of IAV from infected cells, inhibitors of the processing proteases and chemical agents that augment mucosal immunity and/or levels of the relevant defensive compounds may also ultimately prove to be useful as new anti-influenza agents.     

Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines

The oxidative modification of low-density lipoprotein (LDL) is involved in atherogenesis. Among a variety of modified LDLs mentioned in the literature, so-called minimally modified LDL (MM-LDL) was reported to have pro-atherogenic properties despite minimal changes in its oxidative measures. After treatment of LDL with 1 micro M FeSO(4) at 4 degrees C for 96 h, the resulting MM-LDL showed a slight increase in thiobarbituric acid-reactive substances (TBARS) and little association with macrophages. On the other hand, heavily oxidized LDL, which was prepared by copper-induced oxidation of LDL at 37 degrees C, showed a sharp increase in TBARS and strong association with macrophages. By introducing a fluorometric procedure to detect aldehyde-containing phosphatidylcholines (aldehyde-PCs), we examined the amounts of aldehyde-PCs in modified LDL preparations. Aldehyde-PCs increased to 23.4 pmol/ microg protein in MM-LDL, which was more than four-fold higher than in the heavily oxidized LDL. We conclude that MM-LDL is a unique type of oxidized LDL enriched with aldehyde-PCs.     

Half-life of cholesterol 7alpha-hydroxylase activity and enzyme mass differ in animals and humans when determined by a monoclonal antibody against human cholesterol 7alpha-hydroxylase

A monoclonal antibody against human cholesterol 7alpha-hydroxylase was produced, and the half-life of the enzyme was studied. Both the activity and protein mass of the enzyme were measured at timed intervals during microsomal incubation. The enzyme activity dropped rapidly; the half-life was 1, 1.7 and 3h in humans, rats and rabbits, respectively. In contrast, the protein mass, measured by immunoblotting, declined slowly; its half-life was 5h in the human and 9h in the rat and rabbit enzymes. This suggests that there may be vulnerable sites responsible for the enzyme activity. Addition of dithiothreitol (DTT) restored the decreased activity considerably, indicating that at least one sulfhydryl group is involved in the vulnerability. These results show considerable decrement in cholesterol 7alpha-hydroxylase activity due to sulfhydryl groups.     

CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.     

4-Hydroxybenzyl alcohol accumulates in flowers and developing fruits of carrot and inhibits seed formation

Somatic embryogenesis in carrot (Daucus carota) is autonomously inhibited by 4-hydroxybenzyl alcohol (4HBA), which is produced by embryogenic cells. Because somatic embryogenesis is used as a model of zygotic embryogenesis, we assayed for 4HBA in carrot seeds and analyzed the effect of 4HBA on seed formation to determine whether 4HBA is also produced during zygotic embryogenesis. HPLC analysis showed that 4HBA accumulated in flowers and immature and mature fruits, but not in vegetative tissues. The concentration of 4HBA was highest after flowering, when the zygote developed into the early globular-stage embryo. 4HBA accumulation then decreased with seed development. Exogenous application of 4HBA to immature carrot fruits inhibited seed formation. Many 4HBA-treated seeds did not include a mature embryo. These results indicate that the production and accumulation of 4HBA occurs during carrot seed development and that 4HBA has an inhibitory effect on carrot seed formation.     

Preparation of radioactive aldehyde-containing phosphatidylcholine

Short-chain, aldehyde-containing phosphatidylcholine (PC), formed during the oxidation of PC, is thought to be involved in cellular responses in atherosclerosis and inflammation. Here we report a convenient procedure for a small-scale preparation of aldehyde-containing PC. PC containing an unsaturated fatty acyl chain was treated with osmium tetroxide followed by sodium periodate at room temperature. The reaction product was purified by TLC. This preparation showed a single peak on reverse-phase HPLC, and its identity was confirmed by fast atom bombardment mass spectrometry. This procedure does not require special equipment and is easily applicable for preparation of radioactive materials.