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C S Young

Publications and source records attributed to C S Young.

At least 37 records · Page 2Linked to original sources
PubMed1997

Functional characterization of the major late promoter of mouse adenovirus type 1.

During the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mouse adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenced and compared to that of the human virus MLP. The general organization of the transcriptional elements of the putative MAV-1 MLP is similar to that of the human virus counterpart, with some interesting differences. We wished to investigate the function of the predicted MLP of MAV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both Northern blot and primer extension analyses were performed on intracellular RNA isolated from cells infected with MAV-1. The results show that late RNA can be detected 48 hr postinfection and increases up to 6 days p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue of the predicted TATA box. To analyze the functional significance of the predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequence, was established. The predicted MLP sequence was capable of directing luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 site, and the TATA box, either singly or in combination. Each single-element mutation causes a marked reduction in luciferase gene expression, with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1-binding site was examined by gel mobility shift assay and shown to interact with purified SP1 protein specifically, supporting the functional significance of this transcriptional element. These findings contribute to a better understanding of gene expression in MAV-1 and to its development as an appropriate model for the study of the molecular basis of pathogenesis in a natural host animal.

Adenoviridae↗
PubMed1997

The initiator element of the adenovirus major late promoter has an important role in transcription initiation in vivo.

Previous results showed that the structure and function of the adenovirus major late promoter (MLP) can be analyzed genetically in its correct location, despite its essential role in the viral life cycle. This genetic approach was extended to investigate the in vivo role of the initiator (INR), a transcriptional element that surrounds the start site of transcription. The analysis was designed to investigate if the INR is an alternative basal element to the canonical TATA box of the MLP, its relative importance in the functioning of the promoter, and if its function was affected by upstream activating elements. Accordingly, two different mutations in the INR were created and tested in the genome, either by themselves or together with mutations in the TATA box or one of the two upstream activating elements, the upstream promoter element (UPE) and the inverted CAAT box. The mutant viruses were examined first in one-step growth experiments, and then levels of late mRNA accumulation were measured by primer extension, transcription initiation was assayed in isolated nuclei, and viral DNA accumulation was determined by Southern hybridization. Neither mutation in the INR alone had any discernible phenotypic effects but when coupled to a phenotypically silent mutation in the TATA box gave rise to viruses with growth defects that were attributable to a significantly lowered rate of transcription initiation from the MLP. These results suggest that the INR plays a role in vivo and can act as an alternative basal element in the absence of a functioning TATA box. A virus with mutations in both the INR and the UPE, although viable, likewise had a severe deficiency in transcription, suggesting that the function of the INR is affected by that of the UPE. This contrasts with the previous report that a TATA box-UPE double mutation is not recoverable in virus. In addition, the virus with mutations in both the INR and the inverted CAAT box was phenotypically wild type, unlike the previously described TATA box-CAAT box double mutant, which had a severe transcription deficiency. Taken together, the present and previous genetic results can be interpreted as evidence that in the MLP, the TATA box and the UPE are the more important of the two basal and activating elements, respectively, but that the INR and CAAT can function in transcription initiation. We consider the role of the INR in the formation of the preinitiation complex and speculate on possible protein-protein interactions.

Adenoviridae↗
PubMed1996

The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.

Animals↗
PubMed1996

Conservation of DNA sequence in the predicted major late promoter regions of selected mastadenoviruses.

The major late promoter (MLP) of the subgroup C human adenoviruses is a preeminent model for the study of the mechanisms of basal and activated transcription, both in vivo and in vitro. However, while the structure and function of the human virus MLP has been the subject of extensive investigation, the conservation of the various promoter elements among the adenoviruses from different species has not been examined. Conservation of specific elements would strongly suggest the importance and universality of their function. To address this issue, sequences were obtained from cloned DNAs of several representative Mastadenoviridae, mouse adenovirus type 1 (MAV-1), Tupaia adenovirus type 1 (TAV-1), and two bovine adenoviruses of two distinct subgroups, BAV-3 and BAV-7. The results of the sequencing studies showed that the TATA box and an upstream inverted CAAT box are conserved in all species and that the binding site for transcription factor USF is present in all except MAV-1, in which a sequence similar to an Sp1-binding site is present at a similar position. The initiator element (INR) sequence is not well conserved, and only one or other of the two downstream activating elements, DE1 and DE2, is predicted to be present in the nonprimate virus MLP regions. Ribonuclease protection assays on RNA isolated from MAV-1-infected cells late in infection indicated that the predicted MLP is functional, and transcription initiation and splice donor sites were identified. The human virus MLP is embedded in the essential DNA polymerase sequence on the opposite DNA strand. The primary amino acid sequences of the C-terminal regions of the predicted DNA polymerases show strong conservation of sequence motifs observed in replicative polymerases ranging from prokaryotes to mammals, and additional regions of strong conservation among the adenovirus polymerases. Pairwise comparisons between the newly sequenced regions of the polymerases and previously published sequences show that BAV-7 is most dissimilar to all others, while TAV-1 has a greater similarity to the primate sequences than to the others. The sequence data from both strands were also used to construct phylogenetic trees, based on BAV-7 as the outgroup. The trees constructed from the two sets of sequences are broadly similar, showing close relationships between primate viruses, but differing in the order of divergence of TAV-1 and MAV-1 branches.

Animals↗
PubMed1996

Prospective evaluation of fluorine-18 fluorodeoxyclucose positron emission tomography in breast cancer for staging of the axilla related to surgery and immunocytochemistry.

The noninvasive staging of axillary lymph nodes for metastases is investigated in patients with breast cancer prior to surgery by positron emission tomography (PET) with fluorine-18-fluoro-2-deoxy-d-glucose (18F-FDG). In 124 patients with newly diagnosed breast cancer, whole-body PET was performed to determine the average differential uptake ratio (DUR) of 18F-FDG in the axillary lymph nodes. Results were correlated with the number of the dissected lymph nodes, size of the primary tumor, tumor type, tumor grade, estrogen and progesterone receptors, DNA ploidy, and the proportion of cells in the synthetic phase of the cell cycle (S-phase). In this prospective study of 124 patients with breast carcinoma, PET correctly categorized all 44 tumor-positive axillary lymph nodes, a sensitivity of 100%. Sixty tumor-negative axillary lymph nodes were negative by PET and 20 tumor-negative axillary lymph nodes were positive by PET. No false-negative PET findings were encountered. A weak correlation was found between DUR and tumor size as well as between DUR and the S-phase of the tumor. In patients with breast carcinoma, 18F-FDG PET can be of value in evaluating axillary lymph nodes for metastatic involvement prior to surgery. It is of particular importance that no false-negative PET findings were encountered, and axillary lymph node dissection might not be necessary in patients without axillary uptake by PET. The DUR of the positive axillary lymph nodes seems to bear a relationship with some of the purported prognostic parameters of the primary tumor.

Axilla↗
PubMed1996

Increased regional cerebral glucose metabolism and semantic memory performance in Alzheimer's disease: a pilot double blind transdermal nicotine positron emission tomography study.

Nicotinic receptor dysfunction and impaired semantic memory occur early in Alzheimer's disease patients (AD). Previous research implied that nicotine's ability to enhance alertness, arousal, and cognition in a number of nonclinical populations was a function of its ability to stimulate CNS nicotinic cholinergic receptors. In this study it was hypothesized that transdermal administration of nicotine would increase both regional cerebral glucose metabolism (rCMRglc) and semantic memory (as assessed by verbal fluency). Two mild AD and two elderly controls underwent positron emission tomography scanning during a double blind nicotinic agonist verbal fluency challenge procedure. rCMRglc increases occurred in both AD patients, but not controls. In the two AD patients, verbal fluency scores increased by an average of 17%. One elderly control's verbal fluency increased, and the other decreased. These findings suggest that nicotine's effect on metabolism and verbal fluency is due to its ability to stimulate the cholinergic system.

Alzheimer Disease↗
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↗