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Biomedical subjects

C S Young

Publications and source records attributed to C S Young.

At least 19 recordsLinked to original sources
PubMed1995

A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts.

The joining of DNA double-strand breaks in vivo is frequently accompanied by the loss of a few nucleotides at the junction between the interacting partners. In vitro systems mimic this loss and, on detailed analysis, have suggested two models for the mechanism of end-joining. One invokes the use of extensive homologous side-by-side alignment of the partners prior to joining, while the other proposes the use of small regions of homology located at or near the terminus of the interacting molecules. to discriminate between these two models, assays were conducted both in vitro and in vivo with specially designed substrates. In vitro, molecules with limited terminal homology were capable of joining, but analysis of the junctions suggested that the mechanism employed the limited homology available. In vivo, the substrates with no extensive homology end-joined with equal efficiency to those with extensive homology in two different topological arrangements. Taken together, these results suggest that extensive homology is not a prerequisite for efficient end-joining, but that small homologies close to the terminus are used preferentially, as predicted by the modified single-strand annealing model.

Base Sequence↗
PubMed1995

Sequence of the mouse adenovirus serotype-1 DNA encoding the precursor to capsid protein VI.

The nucleotide sequence predicted to encode the precursor to virion structural protein VI (preVI) of mouse adenovirus (Ad) serotype-1 (MAV-1) was determined. The 237-amino-acid sequence has 45% identity and 66% similarity to the human Ad serotype-2 preVI sequence. There is a marked conservation at the C terminus, the last eleven residues of which may be necessary for activating the Ad endoproteinase, and at the N terminus, including the consensus endoproteinase cleavage site.

Amino Acid Sequence↗
PubMed1995

Frameless stereotaxy for radiosurgical planning and follow-up.

In our centre, 111 patients have been treated with linear accelerator stereotactic radiosurgery. Angiographic, CT and MRI images are generated and the target coordinates calculated in 3 dimensions. For CT scanning, cross sections of perpendicular and oblique fiducial markers are seen. For follow-up CT scans done without the frame, a virtual frame is generated by means of a computer program that places fiducial markers on each CT scan cut, as if the patient had been wearing the OBT frame and the scan produced with the gantry parallel to the base of the frame. The position of the oblique marker may be calculated by knowing the thickness and position of each CT cut. Various natural fiducial markers (bony landmarks) are identified by coordinates in the scan with the patient wearing the real frame and in the scan with the virtual frame applied. A transformation matrix is utilized to establish the equivalence between the original CT scan with the real frame applied and subsequent scans without the real frame but with the virtual frame applied. In effect, the virtual frame is re-applied in exactly the same position as the real frame. Lesion measurements may then be duplicated and growth or regression accurately established. The uncertainty in this system of re-application resides in possible patient movement, CT scan slice thickness and inter-observer error in the identification of natural fiducial markers.

Brain Neoplasms↗
PubMed1995

Stereotactic radiotherapy for AVMs: the University of Toronto experience.

Since July 1989, 66 patients have received stereotactic radiosurgery for arteriovenous malformations of the brain. All cases were reviewed by our multidisciplinary group. As result of our treatment algorithms these patients underwent stereotactic radiosurgery, either as the sole therapy or as part of combined modality treatment. Using a 6 MV linear accelerator, we have usually employed doses of either 15 or 20 Gy to the edge of the lesion, ensuring that critical normal structures do not receive a dose in excess of 15 Gy. Of the initial 24 patients followed for a minimum of 2 years, 12 have complete obliteration documented by angiography; 8 have > 90% obliteration (several have deferred further angiographic follow-up which may show progression to complete obliteration); 3 have had the nidus diminish; and one has had no change. Within this cohort, one patient experienced a transient acute effect; one patient has developed a minor late effect; one suffered a fatal hemorrhage despite ongoing response to radiosurgery; one has recently undergone retreatment.

Adult↗
PubMed1995

Implementation of multiple isocentre treatment for dynamic radiosurgery.

Radiosurgery using the dynamic rotation technique with a single isocentre was introduced at the Toronto-Bayview Regional Cancer Centre (T-BRCC) in 1988. Since then, over 100 patients have been treated. It was soon recognized that 25-30% of patients were referred with either non-spherical lesions or multiple lesions located sufficiently close together that consideration had to be given to the overlapping dose distributions throughout the treated volume. To treat these more complex targets a multiple isocentre technique was developed which also took account of these effects and the resulting normalization problem. This technique was implemented in September 1992. Comparisons between calculated doses and actual doses delivered have been undertaken using a spherical phantom containing radiochromic film. Measured dose distributions agreed with the planned distributions to within +/- 1 mm. The effect of multiple isocentres on the penumbra of dose distributions has been examined. The methods adopted for the normalization of treatment plans and clinical examples illustrating the application of the multiple isocentre technique are presented.

Adolescent↗
PubMed1994

Frameless stereotaxy for pre-treatment planning and post-treatment evaluation of radiosurgery.

In our centre, 111 patients have been treated with linear accelerator stereotactic radiosurgery. Angiographic, CT and MRI images are generated and the target coordinates calculated in 3 dimensions. For CT scanning, cross sections of perpendicular and oblique fiducial markers are seen. For follow-up CT scans done without the frame, a virtual frame is generated by means of a computer program that places fiducial markers on each CT scan cut, as if the patient had been wearing the OBT frame and the scan produced with the gantry parallel to the base of the frame. The position of the oblique marker may be calculated by knowing the thickness and position of each CT cut. Various natural fiducial markers (bony landmarks) are identified by coordinates in the scan with the patient wearing the real frame and in the scan with the virtual frame applied. A transformation matrix is utilized to establish the equivalence between the original CT scan with the real frame applied and subsequent scans without the real frame but with the virtual frame applied. In effect, the virtual frame is re-applied in exactly the same position as the real frame. Lesion measurements may then be duplicated and growth or regression accurately established. The uncertainty in this system of re-application residues in possible patient movement, CT scan slice thickness and inter-observer error in the identification of natural fiducial markers.

Brain Neoplasms↗
PubMed1994

Nonhomologous recombination in human cells.

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

Adenoviridae↗
PubMed1994

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

Animals↗
PubMed1994

Neuroleptic medication for dystonia. Reciprocal relationship between effects on motor function and mood.

BACKGROUND: The presence of dysphoric symptoms associated with neuroleptic medication is commonly reported. METHOD: Neuroleptic treatment of a segmental dystonia resulted in a disabling depressive illness, which when treated with antidepressants led to the return of the involuntary movements. RESULTS: The use of several different antidepressants and neuroleptics confirmed the association between drugs and symptoms. Genetic studies excluded "poor metaboliser status" as a reason for the patient's sensitivity to these drugs. CONCLUSIONS: The case illustrates a delicate imbalance between dose of neuroleptic medication and depressive symptoms. We should be aware of the possibility of mood changes in patients treated with neuroleptics for a variety of conditions.

Adult↗
PubMed1993

Genetic analysis of carcinogen enhancement of type 5 adenovirus transformation of cloned Fischer rat embryo fibroblast cells.

Pretreatment of CREF cells with methyl methanesulfonate (MMS) before infection with the host-range cold-sensitive type 5 adenovirus (Ad5) mutant H5hr1 results in a dose-dependent carcinogen enhancement of viral transformation (CET). The properties of CET observed with H5hr1, which include both an MMS dose-dependent enhancement in the number of transformed foci and an increase in transformation frequency after correction for cell toxicity, are not observed in carcinogen-pretreated wild-type (wt) Ad5 (H5wt)-infected CREF cells. This study was conducted to determine the role of the viral E1A and E1B transforming genes of H5hr1 in mediating the unique CET phenotype of H5hr1. Coinfection of MMS-pretreated CREF cells with H5wt or H5sub309 (which displays a wt Ad5 phenotype) and H5hr1 resulted in a suppression of the unique CET phenotype that was directly related to the multiplicity of infection with wt Ad5. Suppression of the unique H5hr1 CET phenotype was also apparent in MMS-pretreated CREF cells coinfected with H5hr1 and an Ad5 mutant expressing either a wt 13S E1A-encoded 289 amino-acid (aa) protein and an intact wt E1B gene or a wt 13S E1A-encoded 289-aa protein and a 22S E1B-encoded 495-aa protein. In contrast, the unique H5hr1 CET phenotype was not suppressed in MMS-pretreated CREF cells coinfected with H5hr1 and Ad5 or Ad2 mutants expressing either a wt 12S E1A-encoded 243-aa protein and both wt E1B gene products or an intact wt E1A gene and a wt E1B 13S-encoded 175-aa protein. That genetic changes in both the E1A and E1B viral regions of H5hr1 were required to induce the unique CET phenotype was also indicated by the inability of a recombinant Ad5 containing the 0-4.5 map-unit region of H5hr1 and the 4.5-100 map-unit region of H5sub309 to display the H5hr1 unique CET phenotype. Direct confirmation of the requirement for both gene regions of H5hr1 to mediate its unique CET was obtained by generating CREF cells stably expressing a wt Ad5 E1A 13S-encoded 289-aa protein and a wt E1B 22S-encoded 495-aa protein. In these CREF transformants (which displayed a CREF-like morphology), transformation by H5hr1 was not reduced, but the unique CET phenotype after MMS pretreatment was eliminated. These results suggest that alterations in both the 13S-encoded E1A and 22S-encoded E1B gene products of H5hr1 contribute to its unique CET.

Adenoviridae↗
PubMed1991

Transcription from the adenovirus major late promoter uses redundant activating elements.

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.

Adenoviridae↗
PubMed1991

End-joining of DNA fragments in adenovirus transfection of human cells.

Overlapping terminal fragments of adenovirus DNA transfected into human cells either recombine to form standard unit-length genomes, or can join end-to-end to produce internally redundant, viable, genomes. The end-joining reaction in human HeLa and A549 cells is almost as efficient as the recombination reaction, and is relatively insensitive to the nature of the ends, as pairs of fragments terminating in several different single strands or in blunt ends can join. In contrast to the results from transfection with SV40, the ends are usually modified, for example by the loss of 3' single strands or the repair of 5' single strands. The ability to recover viable redundant molecules is not confined to any one area of the adenovirus genome, but can occur in the E1 and L2 regions as well as in the E2b region. The redundant genomes contain extra splice signals and may have the capacity to encode fusion proteins.

Adenovirus Early Proteins↗
PubMed1991

Adenovirus homologous recombination does not require expression of the immediate-early E1a gene.

To investigate whether early genes other than those involved directly in DNA replication are required for efficient adenovirus recombination, pairs of viruses with deletions in E1a, E1b 496R, E1b 196R, or E4 and containing differing restriction site markers were used to infect both permissive and non- or semipermissive cells. Recombination was assayed among intracellular and extracellular genomes by restriction digestion and blot hybridization. Recombination was delayed in infections of nonpermissive cells with E1a- viruses until a time consistent with the late onset of DNA replication characteristic of the cell type. This shows that E1a expression is not absolutely required for adenovirus recombination. Similar tests with deletion mutations in E1b and E4 also show that these genes are not required for efficient recombination. Taken together with earlier results showing that recombination depends on DNA replication, it is likely that adenovirus recombination is a consequence of cellular repair functions acting on the substrates produced by replication.

Adenoviridae↗
PubMed1990

Transport and lairage times of lambs slaughtered commercially in the south of England.

The times spent in transport and lairage by 124,036 lambs slaughtered in two plants in the south of England were recorded. About half of them travelled for up to 120 km, taking up to four hours, and three-quarters travelled not more than 300 km, the journeys taking up to about six hours. Bimodal frequency distributions of lairage time were apparent; overall one third of the lambs spent up to four hours in lairage but more than 40 per cent were kept for longer than 14 hours, while they were held overnight. Early season lambs tended to travel shorter distances to slaughter and spent less time in lairage than late season lambs. The average total times spent in transit and lairage were 22.3 hours and 13.3 hours at the two plants.

Abattoirs↗
PubMed1990

Physical mapping of two temperature-sensitive adenovirus mutants affected in the DNA polymerase and DNA binding protein.

We have determined the exact nature of two thermosensitive (ts) adenovirus mutants, H5ts19 and H5ts149, which map to different genes in the E2 transcription unit. The H5ts19 mutation appears to stem from a single base-pair change of A-T to G-C at position 1840 (numbering as in ref. 1), corresponding to codon 154 of the gene coding for DBP. This results in a glutamine-to-arginine change in the amino-terminal domain of the protein. H5ts19 is defective in a late stage of infection, during virus assembly. This phenotype strongly differs from that described for the limited number of known DBP mutants, indicating that DBP is not only functional during DNA replication, but also plays a role in the late phase of the infection cycle. The defect of the (N group) mutant H5ts149 affects the initiation of viral DNA replication. Marker rescue experiments followed by nucleotide sequence analysis of H5ts149 DNA revealed a single point mutation in the gene coding for the Ad pol. A transition of C-G to A-T at position 7563 (numbering as in ref. 2) changes amino acid residue 411 of Ad pol, a leucine residue, to phenylalanine. This mutation is located in a region conserved among various DNA polymerases, which suggests an important role of this domain in DNA replication.

Adenoviridae↗
PubMed1990

Reductive biotransformations of organic compounds by cells or enzymes of yeast.

Saccharomyces cerevisiae catalyses the asymmetric reductive biotransformation of a variety of compounds containing a carbonyl group or carbon-carbon double bond. Oxidoreductases participating in these reactions which have commercial potential in biotransformation processes are likely to have relatively broad substrate specificity. Important carbonyl reductases falling into this category include YADH- and yeast NADP-dependent beta-ketoester reductases. The enoyl reductase component of the FAS complex may have a role in asymmetric yeast reduction of carbon-carbon double bonds of unnatural substrates. Other nicotinamide-requiring oxidoreductases of yeast are also surveyed to rationalize observed biotransformations of whole yeast cells in terms of specific enzymes. Genetic and protein engineering may enable enzymes to be tailored to accept new substrates. A greater understanding of the enzymes and reactions involved will facilitate further optimization and exploitation of these catalytic systems in industrial processes.

Biotransformation↗
PubMed1990

The upstream factor-binding site is not essential for activation of transcription from the adenovirus major late promoter.

An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.

Adenoviridae↗