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.     

Osteogenesis in cultures of limb mesenchymal cells

The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 10⁶ cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 10⁶ cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.     

Plague Meningitis—A Retrospective Analysis of Cases Reported in the United States, 1970-1979

Meningitis caused by Yersinia pestis developed in 6 (6%) of a total of 105 patients with plague reported to the Centers for Disease Control from 1970 to 1979. Five of the six cases occurred in children aged 10 to 15 years. All six patients received antibiotic therapy before meningitis developed, which appeared between the 9th and 14th days after the onset of acute illness in five of the six patients. There were no neurologic sequelae. The antigenic and biochemical profiles of the Y pestis strains isolated from cerebrospinal fluid in the meningitis cases did not differ from those of the Y pestis strains obtained from blood and bubo aspirates in the other 99 patients, and neither did in vitro studies suggest antibiotic resistance. While plague meningitis is an uncommon complication of acute plague infection, physicians in the western United States should be aware that it may develop as much as 14 days after antibiotic therapy for the acute plague infection has been initiated.