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.     

Mitochondrial DNA of the extinct quagga: Relatedness and extent of postmortem change

Sequences are reported for portions of two mitochondrial genes from a domestic horse and a plains zebra and compared to those published for a quagga and a mountain zebra. The extinct quagga and plains zebra sequences are identical at all silent sites, whereas the horse sequence differs from both of them by 11 silent substitutions. Postmortem changes in quagga DNA may account for the two coding substitutions between the quagga and plains zebra sequences. The hypothesis that the closest relative of the quagga is the domestic horse receives no support from these data. From the extent of sequence divergence between horse and zebra mitochondrial DNAs (mtDNAs), as well as from information about the fossil record, we estimate that the mean rate of mtDNA divergence in Equus is similar to that in other mammals, i.e., roughly 2% per million years.     

Responses against antigens encoded by theH-3 histocompatibility locus: Antigens stimulating class I MHC- and class II MHC-restricted T cells are encoded by separate genes

The purpose of this work was to study the genetic basis of histocompatibility antigens encoded by the mouse minor histocompatibility (H) locusH-3. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted helper T cells (TH) specific for antigens encoded by genes within theH-3 locus were isolated and analyzed. Typing a number of mouse strains for expression of antigens recognized by these TH and CTL suggested that there was a different strain distribution pattern of expression of the antigens recognized by TH compared with those recognized by CTL. Separation of the genes whose products stimulate TH from those whose products stimulate CTL was suggested by: (1) analysis of the strain B10.FS(92NX)/Grf that has undergone recombination within theH-3 region; (2) genetic segregation studies of (B10.UW-H-3 ^b/Sn×C57BL/10Sn)F₂ mice; and (3) F₁ complementation studies in which CTL specific for products of the TH-defined gene(s) could not be detected, even in the absence of immune responses to products of the CTL-defined genes. Taken together, these data suggest that in addition to two genes (B2m andCd-1) within theH-3 region whose products typically stimulate class I MHC-restricted CTL, there is at least one additional gene whose product selectively stimulates class II MHC-restricted TH. This new gene is located telomeric from the CTL-defined genes and between the lociwe andun on chromosome 2. These data demonstrate a novel degree of complexity of theH-3 “locus” and suggest selective presentation of minor H gene products in the context of class I or class II MHC proteins.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.