Biomedical subjects

T Nyholm

Publications and source records attributed to T Nyholm.

6 recordsLinked to original sources

PubMed2006

Modelling lateral beam quality variations in pencil kernel based photon dose calculations.

Standard treatment machines for external radiotherapy are designed to yield flat dose distributions at a representative treatment depth. The common method to reach this goal is to use a flattening filter to decrease the fluence in the centre of the beam. A side effect of this filtering is that the average energy of the beam is generally lower at a distance from the central axis, a phenomenon commonly referred to as off-axis softening. The off-axis softening results in a relative change in beam quality that is almost independent of machine brand and model. Central axis dose calculations using pencil beam kernels show no drastic loss in accuracy when the off-axis beam quality variations are neglected. However, for dose calculated at off-axis positions the effect should be considered, otherwise errors of several per cent can be introduced. This work proposes a method to explicitly include the effect of off-axis softening in pencil kernel based photon dose calculations for arbitrary positions in a radiation field. Variations of pencil kernel values are modelled through a generic relation between half value layer (HVL) thickness and off-axis position for standard treatment machines. The pencil kernel integration for dose calculation is performed through sampling of energy fluence and beam quality in sectors of concentric circles around the calculation point. The method is fully based on generic data and therefore does not require any specific measurements for characterization of the off-axis softening effect, provided that the machine performance is in agreement with the assumed HVL variations. The model is verified versus profile measurements at different depths and through a model self-consistency check, using the dose calculation model to estimate HVL values at off-axis positions. A comparison between calculated and measured profiles at different depths showed a maximum relative error of 4% without explicit modelling of off-axis softening. The maximum relative error was reduced to 1% when the off-axis softening was accounted for in the calculations.

Computer Simulation↗

PubMed2000

Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor.

Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.

Base Pairing↗

PubMed1996

A novel ribozyme target site located in the HIV-1 nef open reading frame.

We have tested the sequence UUC CAG UCA GAC CU, at position 9016--9029 within the HIV-1(SF2) nef open reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate-modified 14-nt DNA oligo (complementary to the nef9016--9029 site). A dose-dependent repression of HIV-1(SF2) growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for the nef9016--9029 site (hhRz.nef9016--9029) was constructed and transfected into the human T-cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016--9029 were infected with HIV-1(SF2). A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (10(3)-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviral pol and gag sequences in hhRz.nef9016--9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since no pol and gag PCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support the nef9016--9029 site as a strong new candidate for ribozymal gene therapy against HIV-1 infection.

Base Sequence↗

PubMed1995

A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

Carbon Isotopes↗

PubMed1994

Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels.

3T3 Cells↗

PubMed1993

An avian hepatoma cell line for the cultivation of infectious laryngotracheitis virus and for the expression of foreign genes with a mammalian promoter.

Infectious laryngotracheitis virus (ILTV) is the causative agent of a highly infectious upper respiratory tract disease in chickens. Vaccine development and basic studies on ILTV have been hampered by the lack of a cell line for the cultivation of this herpesvirus which was identified in 1930. Four different avian cell lines were tested for their suitability to propagate ILTV. Here we report the successful growth of ILTV with a chemically-induced avian hepatoma cell line, while retrovirus transformed cell lines derived from permissive primary cells, were found to be non-permissive for ILTV. After multiple passaging of ILTV in the hepatoma cells, the virus could be grown up to a titre of 1 x 10(7) EID50 per ml with a replication cycle comparable to that in primary hepatocytes. Methods of plaque assay, DNA-transfection, and expression of a reporter gene were established. The gene coding for the bacterial beta-galactosidase gene under the control of the cytomegalovirus (CMV) immediate-early promotor was transiently expressed, indicating that a mammalian herpesvirus promotor was recognized by this avian cell line. Infectious ILTV virions were produced after transfecting this cell-line with purified ILTV DNA. The results indicated that the cell line is suitable for the construction of recombinant ILTV and for the molecular biological study of this important avian pathogen.

Animals↗