Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus.

Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.     

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.     

Immunofluorescent localization ofSchistosoma mansoni circulating cathodic antigen in tissues of infected mice using monoclonal antibody

In the present study the kinetics of the uptake and deposition of the major circulating cathodic antigen (CCA) ofSchistosoma mansoni in liver, spleen, and kidney ofS. mansoni infected Swiss mice was investigated in relation to the duration of infection and infection dose (50, 100, 200 cercariae). The presence of antigen was studied with a direct immunofluorescence reaction on frozen sections of the mouse organs, using a fluorescein isothiocyanate (FITC)-labelled mouse IgM monoclonal antibody recognizing a repeating epitope of CCA.CCA was demonstrable from 2 weeks post infection (p.i.) onwards in Kupffer cells in the liver, from 3–4 weeks p.i. onwards in macrophages in the marginal zones in the spleen and from 8 weeks p.i. onwards in kidney glomeruli. The immunofluorescence reactions on CCA in kidney glomeruli, however, remained relatively weak.     

Appearance of extrachromosomal circular DNAs during in vivo and in vitro ageing of mammalian cells

Appearance of extrachromosomal circular DNAs with in vivo and in vitro cellular ageing was examined by using a new technique of mica-press-adsorption for electron microscopy. The size distribution and the copy number of circular DNA complexes varied, depending on the cellular age. Extrachromosomal circular DNA complexes of variable length of more than 0.5 microns or 1.5 kilobase (kb) appeared during in vivo ageing of rat lymphocytes and in vitro ageing of cultured human lung fibroblasts. A restricted size class of circular forms of less than 0.5 microns in contour length was amplified in human skin fibroblasts from aged normal or Werner's syndrome subjects. These circular DNA molecules are suggested to be products of DNA rearrangements or gene amplification occurring in the chromosome.     

Neutron dosimetry in high energy X-ray beams of medical accelerators.

High energy X-ray beams from medical accelerators are used in cancer therapy. In such beams, neutrons are also produced due to photon-neutron interactions in the target material, collimator and beam flattening filter. The feasibility of employing fast-neutron-induced recoil particle tracks in polycarbonate foils developed by the electrochemical etching (ECE) method for the dosimetry of such neutrons was investigated. The experiments were carried out in, and out of, the beam of a Varian Clinac-18, an Allis-Chalmers 25 MeV betatron and a Brown Boveri 45 MeV betatron. Neutron dose equivalents were found both as a function of the photon dose and of the distance away from the centre of the beam. The neutron dose equivalent was found to be 0.6 and 1.3% of the X-ray dose at the centre position of the beam for a TSD = 100 cm and a 10 x 10 cm2 field for the 25 and 45 MeV betatrons respectively. Although the dosemeter showed adequate sensitivity to neutrons in the beam of the betatrons, it showed insensitivity to neutrons in the beam of the Clinac-18 which has a softer spectrum than the neutron energy threshold of the dosemeter. It can be concluded that this dosimetry method provides a new approach with a number of advantages compared to some other existing techniques for such measurements above the energy threshold of the dosemeter.