Enzyme distributions in subcellular fractions of BHK cells infected with Semliki forest virus: evidence for a major fraction of sphingomyelin synthase in the trans-golgi network.

BHK cells either untreated or infected with Semliki Forest virus have been fractionated on sucrose density gradients. Virus infection caused an increase in density of a membrane fraction enriched in sphingomyelin (SM), cholesterol, SM synthase and sialyltransferase activity. This increase in density was related to incorporation of viral proteins into this fraction, which is likely to contain trans-Golgi network (TGN) membranes. In contrast, glucosylceramide synthase and galactosyltransferase activities (markers for cis/medial and trans-Golgi respectively) underwent no density shift and alkaline phosphodiesterase, a plasma membrane marker, was only slightly density-shifted in infected cells. When cells were incubated with NBD-ceramide to enable them to synthesise NBD-SM and then washed with albumin to remove surface label, fluorescence in untreated cells was concentrated in a single juxtanuclear spot but in infected cells this region of bright fluorescence was larger and extended around the nucleus. After fractionation of these cells, NBD-SM (but only a small proportion of the NBD-ceramide) was found to be shifted into the higher density fraction in infected cells. This work provides further evidence that SM synthase is not mainly localised in the early Golgi cisternae as previously thought, but is associated more with a cholesterol-rich compartment which could be the TGN.     

Plating Medium for Differentiating Salmonella arizonae from Other Salmonellae

A differential plating medium developed for isolation of Salmonella arizonae produces a uniform reaction for both lactose-negative and -positive S. arizonae and differentiates S. arizonae from other salmonellae.     

The oncolytic peptide LTX-315 triggers necrotic cell death

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.     

Alterations in the sialylation and sulfation of secreted mouse thyrotropin in primary hypothyroidism

We investigated the effect of in vivo hypothyroidism on the sialylation and sulfation of thyroid-stimulating hormone (TSH) secreted by mouse pituitary explants. Oligosaccharides from secreted thyroid-stimulating hormone from hypothyroid animals contained greater sialic acid relative to sulfate in both alpha and beta subunits. Aging per se had little effect on thyroid-stimulating hormone sialylation or sulfation. Variable sialylation and sulfation demonstrates a mechanism for charge microheterogeneity of thyroid-stimulating hormone, and the increasing sialylation observed with hypothyroidism may functionally mediate the prolonged metabolic clearance that has been noted previously.