Ceramide generated by acidic sphingomyelinase contributes to tumor necrosis factor-α-mediated apoptosis in human colon HT-29 cells through glycosphingolipids formation Possible role of ganglioside GD3

AbstractIn the present study we assessed the contribution of acidic sphingomyelinase (ASMase), a ceramide generating enzyme, in tumor necrosis factor (TNF)-mediated apoptosis in human colon HT-29 cells. TNF induced apoptosis in HT-29 cells in a time- and dose-dependent fashion. Downregulation of the active endogenous ASMase form prevented TNF-stimulated ASMase activity and apoptosis. Furthermore, inhibition of glucosylceramide synthase, which blunted TNF-stimulated GD3 levels, abolished TNF-mediated cell death. Immunocytochemical staining revealed the co-localization of GD3 with mitochondria induced by TNF. The knockdown of targeted GD3 synthase by antisense expression vector protected HT-29 cells against TNF-induced cell death. Thus, ASMase plays a key role in TNF-induced cell death in human colon epithelial cells possibly through GD3 generation

Divergent Reactivity of Mixed-Ligand Manganese(I) Carbonyl–Isocyanide Complexes toward Primary Amines

The reactivity of the mixed-ligand carbonyl–isocyanide complexes <i>fac</i>-[Mn­(CNR)­(CO)₃(bipy)]⁺ (<b>1a</b>, R = Ph; <b>1b</b>, R = xylyl; <b>1c</b>, R = Me; <b>1d</b>, R = CH₂Ph; <b>1e</b>, R = <i>t</i>Bu) with the primary amines NH₂Me and NH₂CH₂CH₂CH₂Br has been studied. Nucleophilic addition of the amine to a carbonyl ligand or to the isocyanide ligand may take place, depending on the nature of the amine, the substituent in the isocyanide ligand, and the reaction conditions; even replacement of a carbonyl ligand may occur. Thus, a variety of carbamoyl (<b>2a</b>–<b>e</b>), diaminocarbene (<b>3a</b>–<b>d</b>, <b>5a</b>–<b>d</b>), N,O-heterocyclic carbene (<b>6b</b>), and amine (<b>4b</b>,<b>c</b>) complexes of manganese­(I) has been obtained and characterized by spectroscopic and/or X-ray diffraction methods

Molecular Characterization of an Endopolygalacturonase from Fusarium oxysporum Expressed during Early Stages of Infection

The tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici produces an array of pectinolytic enzymes that may contribute to penetration and colonization of the host plant. Here we report the isolation of pg5, encoding a novel extracellular endopolygalacturonase (endoPG) that is highly conserved among different formae speciales of F. oxysporum. The putative mature pg5 product has a calculated molecular mass of 35 kDa and a pI of 8.3 and is more closely related to endoPGs from other fungal plant pathogens than to PG1, the major endoPG of F. oxysporum. Overexpression of pg5 in a bacterial heterologous system produced a 35-kDa protein with endoPG activity. Accumulation of pg5 transcript is induced by citrus pectin and d-galacturonic acid and repressed by glucose. As shown by reverse transcription-PCR, pg5 is expressed by F. oxysporum in tomato roots during the initial stages of infection. Targeted inactivation of pg5 has no detectable effect on virulence toward tomato plants