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Biomedical subjects

B R Young

Publications and source records attributed to B R Young.

At least 19 recordsLinked to original sources
PubMed1994

Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC).

A radioresistant cell clone (1B3) was previously isolated after transfection of an ataxia-telangiectasia (AT) group D cell line with a human cosmid library. A cosmid rescued from the integration site in 1B3 contained human DNA from chromosome position 11q23, the same region shown by both genetic linkage and chromosome transfer to contain the genes for AT complementation groups A/B, C, and D. A gene within the cosmid (ATDC) was found to produce mRNAs of different sizes. A cDNA for one of the most abundant mRNAs (3.0 kb) was isolated from a HeLa cell library. In the present study, we sequenced the 3.0-kb cDNA and the surrounding intron DNA in the cosmids. We used polymerase chain reaction, with primers in the introns, to confirm the number of exons and to analyze DNA from AT group D cells for mutations within this gene. Although no mutations were found, we do not rule out the possibility that mutations may be present within the regulatory sequences or coding sequences found in other mRNAs specific for this gene. From the sequence analysis, we found that the ATDC gene product is one of a group of proteins that share multiple zinc finger motifs and an adjacent leucine zipper motif. These proteins have been proposed to form homo- or heterodimers involved in nucleic acid binding, consistent with the fact that many of these proteins appear to be transcriptional regulatory factors involved in carcinogenesis and/or differentiation. The likelihood that the ATDC gene product is involved in transcriptional regulation could explain the pleiomorphic characteristics of AT, including abnormal cell cycle regulation.

Amino Acid Sequence↗
PubMed1994

Expression of the candidate A-T gene ATDC is not detectable in a human cell line with a normal response to ionizing radiation.

Nucleotide sequence analysis of a candidate gene for A-T group D (ATDC) demonstrated that it is related to a group of proteins that contain both zinc finger and leucine zipper motifs. The presence of a leucine zipper suggested that this protein might form homodimers, and this was confirmed by means of the two-hybrid system in yeast. The activity of some proteins that form homodimers can be effectively eliminated by overexpression of inactive forms of the protein that bind to the wild-type protein to create a dominant negative phenotype. An ATDC cDNA containing a 37 amino acid deletion in the zinc finger region (ATDC delta) was therefore transfected into colorectal carcinoma human tumour cells (RKO) to determine whether its expression would produce a response to radiation similar to that seen in A-T cells. RKO cells have been shown to have normal radiosensitivity and cell cycle regulation and, therefore, seemed ideal for this study. Despite the fact that the A-T gene has been found to be important in the radiation damage response, no ATDC mRNA transcripts were detectable in the RKO cell line. In addition, the RKO subclones expressing the ATDC delta mRNA showed no change in radiosensitivity or cell cycle regulation. These results do not support the conclusion that ATDC is an A-T gene, and suggest that the ATDC protein acts indirectly to suppress radiosensitivity in A-T cells.

Amino Acid Sequence↗
PubMed1990

Recombination events during integration of transfected DNA into normal human cells.

The mechanisms of recombination responsible for random integration of transfected DNA into the genome of normal human cells have been investigated by analysis of plasmid-cell DNA junctions. Cell clones containing integrated plasmid sequences were selected by morphological transformation of primary human fibroblasts after transfection with a plasmid containing simian virus 40 sequences. Nucleotide sequence analysis of the plasmid-cell DNA junctions was performed on cloned DNA fragments containing the integration sites from two of these cell clones. Polymerase chain reaction was then performed with human cell DNA from primary fibroblasts to isolate the cell DNA from the same sites before plasmid integration. Comparison of the sequences at the plasmid-cell DNA junctions with those of both the original plasmid and the cell DNA demonstrated short sequence similarities and additional nucleotides, typical of nonhomologous recombination. Evidence of short deletions in the cell DNA at the plasmid integration sites suggests that integration occurred by a mechanism similar to that used for repair of spontaneous or gamma ray-induced strand breaks. Plasmid integration occurred within nonrepetitive cell DNA with no major rearrangements, although rearrangements of the cell DNA at the integration site occurred in one of the clones after integration.

Base Sequence↗
PubMed1989

Nucleotide sequence analysis of novel junctions near an unstable integrated plasmid in human cells.

Characterization of human cell clones containing a promoterless selectable gene (neo), integrated at various locations in the genome, demonstrated that one of the integration sites had a high rate of spontaneous tandem duplications. Other investigators have suggested that specific sequences, such as short repeats, found near an integration site, could be responsible for this kind of instability. To learn more about this process, we sequenced the DNA at the recombination site in two independently derived subclones, and compared these sequences with those found in the parental cell DNA. The results demonstrate that specific sequences are not required at the recombination site. In one G418-resistant subclone, recombination occurred between an Alu retroposon in the cellular DNA and integrated pBR322 sequences sharing 3 bp of similarity at the recombination site. In the other subclone, recombination occurred between single-copy cellular DNA and integrated simian virus 40 sequences sharing a single bp of similarity at the recombination site. This heterogeneity at the recombination site indicates a general enhancement of the rate of recombination within the entire region, with little if any sequence specificity or similarity required.

Base Sequence↗
PubMed1989

Radioresistant DNA synthesis and human genetic diseases.

Sixty-eight human fibroblast cell strains were assayed for radioresistant DNA synthesis (RDS), which is defined here as the absence of a steep component of inhibition of DNA synthesis in a dose-response curve when rate of DNA synthesis is plotted against radiation doses from 0 to 20 Gy or more. Twenty-seven strains from patients who were previously diagnosed to have ataxia-telangiectasia (AT) were positive for this feature. Among the cell strains that did not show RDS were two from AT obligate heterozygotes (i.e., the parents of AT patients), two from patients with Alzheimer disease, two from patients with Friedreich ataxia, one from a patient with Bloom syndrome, one from a patient with Down syndrome, and six from patients with various immunodeficiencies. Four strains demonstrated RDS that was less pronounced than in most AT cells: one was from a patient with Nijmegen breakage syndrome, one was from a patient without ataxia but with choreiform movement disorder, telangiectasia, and elevated concentrations of alpha-fetoprotein in the blood, and two were from AT patients. RDS therefore is not a necessary trait of human genetic diseases that involve radiosensitivity or immunodeficiency. Although recent reports suggest that some AT patients do not exhibit RDS, we found RDS in all the AT cells we tested.

Ataxia Telangiectasia↗
PubMed1988

Protection of mice and swine from pseudorabies virus-induced mortality by administration of pseudorabies virus-specific mouse monoclonal antibodies.

Hybridomas were selected for secretion of monoclonal antibodies directed against pseudorabies virus (PRV) glycoproteins. Each monoclonal antibody was capable of neutralizing PRV in vitro in the presence of complement. This panel of antibodies was used in passive immunization studies to protect mice and swine from PRV-induced mortality. The most protective antibody in mice was 3A4, specific for PRV glycoprotein gp50, which afforded as high as 100% protection. Although antibody 3A4 was partially protective in swine, antibody 3D11, which is specific for PRV glycoprotein glll, afforded greater protection--83% protection when ascitic fluid was used and 100% protection when immunoglobulin concentrated from cell cultures was used at a dose of 150 mg/pig. These studies demonstrated that monoclonal antibodies may be useful for short-term prophylaxis against PRV-induced disease and that antibody directed against either PRV glycoprotein glll or gp50 is sufficient to protect animals from PRV-induced mortality.

Animals↗
PubMed1987

Absence of DNA overreplication in Chinese hamster cells incubated with inhibitors of DNA synthesis.

Previous reports have suggested that transient inhibition of DNA synthesis by chemicals or ultraviolet light causes some of the DNA to replicate more than once in one cell cycle, i.e., that it induces overreplication of DNA. The data that led to this suggestion were obtained from cesium chloride equilibrium density gradient analyses, in which cells were incubated with bromodeoxyuridine so that the DNA synthesized after incubation with the inhibitor could be densitometrically separated from the DNA that had been radioactively labeled before incubation with the inhibitor. An unresolved problem with these analyses was that the data also suggested that overreplication must have occurred in control cells, i.e., those not incubated with an inhibitor of DNA synthesis. We show here that the latter result is probably due to an artifact of cesium chloride equilibrium density gradient analysis, probably because of nonspecific trapping of DNA in regions of the gradients where there are large amounts of DNA. We also used another protocol that avoids this artifact; with this protocol any overreplicated DNA would be found where heavy-heavy DNA bands and nonspecific trapping cannot occur. When this protocol was used there was no evidence that transient inhibition of DNA synthesis induces overreplication of DNA.

Animals↗
PubMed1987

DNA synthesis in irradiated mammalian cells.

One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v.-induced inhibition of replicon initiation.

Animals↗
PubMed1986

The influence of preadsorbed canine von Willebrand factor, fibronectin and fibrinogen on ex vivo artificial surface-induced thrombosis.

We have examined the effects of preadsorption of several canine plasma proteins on surface-induced thrombogenesis in a canine ex vivo model. Our technique allowed determination of initial deposition and subsequent embolization of 51Cr-labeled platelets and 125I-fibrinogen onto and from polymeric arterio-venous shunts in non-anticoagulated canines. Segments of the tubing were removed at various time points between 2 and 120 minutes of blood contact for examination of the morphology of the thrombus by scanning electron microscopy. Thrombus deposition was measured on uncoated plasticized poly(vinyl chloride) (PVC) and PVC precoated with canine von Willebrand factor (vWF), fibronectin, partially purified fibrinogen (fibrinogen which contained vWF and fibronectin as impurities), or purified fibrinogen (fibrinogen which had been further purified to remove fibronectin and vWF). Preadsorption of all proteins studied enhanced the thrombogenic response relative to that of the uncoated surface. Precoating with vWF or partially purified fibrinogen resulted in the deposition of the greatest number of thrombi, and embolization was slower than on shunts precoated with canine fibronectin or purified fibrinogen. The deposition-embolization profiles for the fibronectin and purified fibrinogen-coated surfaces were similar. The amount and time sequence of initial adhesion and spreading of platelets was related to the extent and time sequence of peak thrombus formation. The partially purified fibrinogen-coated and vWF-coated surfaces had more adhered and spread platelets at the earliest time points and a greater number of larger thrombi at the peak deposition times. The slowest rate of platelet adhesion and spreading was seen on the purified fibrinogen-coated surface. White blood cells were present very early on surfaces precoated with vWF and partially purified fibrinogen, and were present prior to embolization on all surfaces. Major conclusions from this work indicate that, although fibrinogen and fibronectin promote thrombogenesis when adsorbed to a surface, vWF is even more active in promoting platelet deposition and in anchoring thrombi to the surface of biomaterials. Thus, differences in vWF adsorption to biomaterials may be a determinant of surface-induced thrombogenesis.

Adsorption↗
PubMed1983

Physicochemical characterization and in vivo blood tolerability of cast and extruded Biomer.

Solution grade and extruded grade Biomer (SB and EB, respectively) are polyurethanes that have been suggested for use in biomedical applications. The bulk materials were examined by elemental analysis, differential scanning calorimetry, thermomechanical testing, and stress-strain testing. The extruded grade material has a lower soft segment molecular weight (650 g/mol) than the solution grade material (2000 g/mol). As a result of its higher molecular weight, the soft segment phase of SB is semicrystalline in the solid state. The hard segments of the extruded grade material are chain extended with water yielding a lower urea concentration than in the solution grade material in which the hard segments are chain extended with diamines. Chemical structures for the two materials consistent with elemental analysis, urea/urethane ratios and thermal and mechanical data, are proposed. X-ray photoelectron spectroscopy (ESCA) was used to analyze the surfaces of extruded grade Biomer, solution grade Biomer cast on the inner surface of polyethylene tubing, and extruded grade Biomer dissolved in DMA and similarly cast on polyethylene (CB). Soft segment concentrations were highest on the EB surface and lowest on the SB surface. Soft segment concentrations on the EB surface were higher than on the CB surface, indicating that the method of fabrication affected the composition of the surface layer. The three materials were tested for blood tolerance in a canine femoral arteriovenous shunt configuration. Blood compatibility was correlated with increasing concentration of polyether soft segments on the surface.

Animals↗
PubMed1981

Cast vs extruded Biomer for biomedical applications.

The properties of solution grade and extrusion grade Biomer have been investigated and compared. In vivo blood compatibility studies using arteriovenous (AV) shunts of the cast and extruded materials show that extruded Biomer is more thromboresistant than solution cast Biomer. This has important implications for long-term implantation studies, since solution cast Biomer is used in artificial hearts. This study also indicates that extruded Biomer, dissolved in DMA and then solution cast, is more thromboresistant than cast solution grade Biomer. Surface studies using ESCA indicate a correlation between thromboresistance and surface soft segment concentration.

Animals↗
PubMed1980

Radiosensitivity in ataxia-telangiectasia: a new explanation.

The cause of increased radiosensitivity in ataxia-telangiectasia (AT) cells may be a defect in their ability to respond to DNA damage rather than a defect in their ability to repair it. Doses of x-radiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition in AT cells and thus less delay during which x-ray damage could be repaired. The radioresistance of DNA synthesis in AT cells was primarily due to a much smaller inhibition of replicon initiation than in normal cells; the AT cells were also more resistant to damage that inhibited chain elongation. AT cells have been reported to undergo less radiation-induced mitotic delay than normal cells, which may cause them to move from G2 phase into mitosis before repair is complete and may result in the increased incidence of chromatid aberrations observed by others. Therefore, AT cells fail to go through those delays that allow normal cells to repair DNA damage before it can be expressed.

Ataxia Telangiectasia↗
PubMed1978

Anomalous effects of 125I after its incorporation into mammalian cell DNA.

During labeling of mammalian cells with 125I-iododeoxyuridine for studies on single-strand break induction and repair, care must be taken to keep the amount of 125I incorporated per cell to very low levels. Under some conditions enough 125I can be incorporated during the incubation period (generally about one generation time) to damage the repair systems of cells so extensively, even before they are frozen, that they cannot repair any of the breaks induced by the 125I during the time they are frozen to accumulate these breaks.

Animals↗
PubMed1976

Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells.

X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.

Cell Line↗