PubMed Health⌕ Search

Biomedical subjects

H Kroath

Publications and source records attributed to H Kroath.

9 recordsLinked to original sources
PubMed1999

Genetically modified organisms in food-screening and specific detection by polymerase chain reaction.

PCR methods for the detection of genetically modified organisms (GMOs) were developed that can be used for screening purposes and for specific detection of glyphosate-tolerant soybean and insect-resistant maize in food. Primers were designed to amplify parts of the 35S promoter derived from Cauliflower Mosaic Virus, the NOS terminator derived from Agrobacterium tumefaciens and the antibiotic marker gene NPTII (neomycin-phosphotransferase II), to allow for general screening of foods. PCR/hybridization protocols were established for the detection of glyphosate-tolerant RoundUp Ready soybean and insect-resistant Bt-maize. Besides hybridization, confirmation of the results using restriction analysis was also possible. The described methods enabled a highly sensitive and specific detection of GMOs and thus provide a useful tool for routine analysis of raw and processed food products.

Blotting, Southern↗
PubMed1982

Interferon inhibits establishment of fibroblast infection with avian retroviruses.

Pretreatment of chick embryo fibroblasts (CEF) with low doses of homologous interferon (16 u/ml) drastically inhibits cell transformation by, and replication of Rous sarcoma virus (RSV). Treatment of chick cells with 16 u/ml of interferon before de novo infection with a transformation defective (td) mutant-RSV, also resulted in a reduction of extracellular virus particles. This was determined by infectivity titrations, virus associated reverse transcriptase (RT) activity and measurement of metabolically radioactively labelled virus particles. The viral proteins pr 180, pr 76, p 27, p 19 and p 12 were still synthesized in interferon-treated cells in an unaltered form, although at slightly reduced levels. No difference in the pattern of structural proteins could be detected between virus particles harvested from cells treated with interferon and from control cells. In contrast to de novo infected cells, concentrations of interferon as high as 200 u/ml had no influence on the reversible transformation of cloned fibroblasts infected with a temperature sensitive mutant of RSV. In addition, fibroblasts infected with td-SR-RSV-D before addition of interferon showed only a marginal effect on formation of infectious virus even after treatment with 200-500 u/ml of interferon. This was not caused by interferon-resistance of the td-SR-RSV-D infected cells since viral protein synthesis by superinfecting Vesicular stomatitis virus (VSV) was as sensitive to interferon as in cells not preinfected with retrovirus. Our results support the notion that exogenous infection of fibroblasts with avian retrovirus is inhibited by interferon during an early phase of the replication cycle.

Animals↗
PubMed1982

mRNA 5'-cap binding activity in purified influenza virus detected by simple, rapid assay.

Reovirus mRNA 5'-terminal caps were 3'-radiolabeled with pCp and as affinity probes for proteins with cap binding activity. A rapid, simple, and sensitive blot assay was devised that could detect cellular cap binding protein in a complex polypeptide mixture. By using this method, cap binding activity was found in detergent-treated influenza virus but not in reovirus or vaccinia virus. Preincubation of capped reovirus mRNA with purified cellular cap binding protein reduced its primer effect on influenza transcriptase, whereas priming by ApG was not affected. The results indicate that influenza transcriptase complexes include cap-recognizing proteins that are involved in the formation of chimeric mRNAs.

Carrier Proteins↗
PubMed1978

Analysis of the methylated 'cap' structures of vaccinia mRNA by two-dimensional thin-layer chromatography.

Two different twodimensional cellulose thinlayer separations for blocked, methylated mRNA 5'-termini are described. They allow rapid analysis even of complex mixtures of mRNA "cap" structures on the basis of their methyl group content and base composition. These simple procedures are especially useful for the analysis of [3H-methyl]-labeled mRNA in combination with tritium fluorography. A qualitative and quantitative analysis of the methylated "cap" structures of in vitro labeled Vaccinia "core" mRNA is presented. The presence of methylated "cap" structures in Vaccinia RNA increases the in vitro translation efficiency of methylated Vaccinia RNA over Vaccinia RNA transcribed in the absence of a methyl group donor.

Chromatography, Thin Layer↗
PubMed1978

RNA methylation in vaccinia virus-infected chick embryo fibroblasts treated with homologous interferon.

Interferon-pretreatment of vaccinia-infected chick embryo fibroblasts resulted in a greater than 50% decrease in ribose methylation of the penultimate "cap" nucleotide in virus-specific mRNA. However, in contrast to results obtained with cell-free systems, in intact infected cells there was (a) no detectable reduction in methylation of the 5'-ultimate m7G of viral mRNA; (b) a virus specificity of the interferon-induced change in mRNA "CAP"-methylation seems unlikely and (c) analysis of the ribosomal and transfer RNA fractions isolated from interferon-treated and control cells revealed identical patterns of methylated nucleotides. Thus, the interferon-induced change in methylation is specific for mRNA "CAPS".

Cells, Cultured↗
PubMed1976

Studies on kinetics of DNA synthesis in CV-1 cell cultures infected with different multiplicities (MOI) of Herpes simplex virus type 2 (HSV-2).

Infection of CV-1 cell cultures with HSV-2 at MOI 5 respectively 1PFU/cell resulted in markedly different patterns of DNA-synthesis. Isolation and separation of cellular and viral DNA on CsCl equilibrium density gradients during the "late" phase after infection with 5PFU/cell revealed a rapid increase in synthesis of virus-specific DNA (which banded at density q25 = 1.729 g/ccm) while synthesis of host cell DNA (banding at 1.705 g/ccm) was constantly inhibited. 15 hours after infection and incubation with labelled medium, around infection and incubation with labelled medium, around 75% of total isolated DNA was virus-specific. On lowering the MOI to 1 PFU/cell, however, synthesis of host cell DNA continued and was even partially stimulated during the late phase whereas synthesis of virus-specific DNA advanced only slowly. 16 hours after infection, approximately 25% of the total assayed after infection, approximately 25% of the total assayed DNA was virus-specific and the amount of host cell DNA approached values of uninfected control cells. 24 hours after infection and incubation, virus specific DNA rose to approximately 45% of overall DNA assayed, and synthesis of host cell DNA was completely inhibited. However, infectivity rose constantly and reached 106.8 TCD 50/ml at 22 hours p.i. These findings were independent of passage number of CV-cell culture, and there were no alterations in karyotype and morphological behaviour of the cells.

Animals↗