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M D Sargent

Publications and source records attributed to M D Sargent.

At least 19 recordsLinked to original sources
PubMed1997

A three-step allele-level DRB1-DRB3-DRB4-DRB5 genotyping assay using polymerase chain reaction with immobilized sequence-specific oligoprobes.

A three-step reverse hybridization assay for allele level-resolution DRB1-DRB3-DRB4-DRB5 genotyping is described. Samples are initially amplified using a generic primer pair for all DRB1-DRB3-DRB4-DRB5 alleles and PCR products are hybridized to generic typing membranes. An intermediate-resolution level genotyping is obtained at this point. Depending on the phenotype, samples are then subjected to a DR1, DR2, DR4, DR52A, DRB3 and/or DRB5 type-specific amplification and hybridization. A third step, involving sequence-specific PCR followed by type-specific hybridization, is only performed to solve certain DR4 and DR52A heterozygous combinations. The assay allows 100% allele level-resolution DRB genotyping. Hybridization membranes contain panels of SSO probes that were optimized to all react specifically under identical stringency conditions. A computer program was written to assist in analysis of the hybridization patterns. The assay was throughly evaluated and has been used to type over 10,000 donors from the Canadian Unrelated Bone Marrow Donor Registry (UBMDR) at allele level-resolution. This method proved to be flexible, easy to update for newly described alleles, easy to perform, fast, and safe. It is also reliable and specific, as 9 novel DRB alleles so far have been detected as aberrant hybridization patterns.

Alleles↗
PubMed1990

Inhibitors of repair of potentially lethal damage and DNA polymerases also influence the recovery of potentially neoplastic transforming damage in C3H10T-1/2 cells.

The effects of aphidicolin and beta Ara A on radiation sensitivity were evaluated in terms of cell killing, recovery, and neoplastic transformation in the C3H10T-1/2 cell system. When cells were held in plateau phase, recovery of potentially lethal damage (PLD) and potentially transforming damage (PTD) occurred. The addition of beta Ara A resulted in reduced PLD recovery for both the survival and neoplastic transformation end points. The addition of aphidicolin did not affect recovery of PLD or PTD. These data show that the inhibition of polymerase alpha by aphidicolin does not affect recovery of damage leading to cell death or neoplastic transformation. However, the inhibition of both polymerase alpha and beta by beta Ara A resulted in inhibition of recovery of damage leading to both cell death and neoplastic transformation. These data indicated that polymerase beta may be involved in both PLD and PTD recovery.

Animals↗
PubMed1988

Enrichment of progenitor cells from human bone marrow by flow cytometry.

Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean +/- standard deviation) with respect to the total was as follows: Region 1: 17.6 +/- 9.9; region 2: 5.3 +/- 1.4; region 3: 71.7 +/- 9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells.

Bone Marrow Cells↗
PubMed1988

Response of human bone marrow progenitor cells to X-rays in vitro.

In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23 degrees C, was characterized by a two-term exponential relationship, with D0 values of 0.42 +/- 0.19 Gy and 1.38 +/- 0.37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D0 = 1.18 +/- 0.15 Gy. The biphasic response at 23 degrees C became a single exponential response when the temperature at irradiation was 30 or 37 degrees C.

Bone Marrow↗
PubMed1987

DNA radiation damage and its modification by metallothionein.

Thiol compounds have long been known to protect living cells against the harmful effects of ionizing radiation. Maetallothionein is a naturally occurring low molecular weight polypeptide rich in cysteine residues and may be useful in protection against low-level radiation effects. Radiation damage to DNA and its nucleotide components and the radioprotective effect of metallothionein have been studied in model chemical systems and compared to its effect on cells. Metallothionein acts both as a free radical scavenger and a reductant, and its radioprotective effectiveness has been studied as a function of dose, drug concentration, and in the presence and absence of oxygen. It is more effective in protecting against sugar-phosphate damage under hypoxic conditions. The chemical modification is greater than that of cell killing as measured by the loss of colony-forming ability. Dose reduction factors greater than two are observed for DNA radioprotection, but the values in cells are much lower. These findings will be discussed in terms of the molecular mechanisms and their implications.

Animals↗
PubMed1986

Selective cytotoxicity of calmidazolium and trifluoperazine for cycling versus noncycling C3H10T1/2 cells in vitro.

Effects of two anticalmodulin drugs, trifluoperazine and calmidazolium, on normal and transformed C3H10T1/2 cells were examined in vitro. As indicated by reduction of plating efficiencies in the presence of these drugs, the intrinsic sensitivities of normal and transformed cells were similar and showed no consistent differences. Comparison of cell killing kinetics in cycling and noncycling cell populations revealed that both drugs were preferentially cytotoxic for cycling cells. This differential cytotoxicity for cycling versus noncycling cells could provide a basis for exploitation of anticalmodulin drugs in cancer chemotherapy.

Animals↗
PubMed1986

Oncogenic transformation of C3H10T1/2 mouse embryo cells by X-rays, hyperthermia, and combined treatments.

The C3H10T1/2 cell system was used to measure oncogenic transformation after heating and after combined treatments of heat and X-rays. Heat alone at 41.0, 42.0, and 45.0 degrees C caused no transformation. Prolonged heating at 42.0 degrees C, or incubation at 37 degrees C after heating at 45.0 degrees C, resulted in thermotolerance in terms of cell killing, but did not cause transformation. Combined treatments of heat and radiation resulted in small increases in the transformation frequency per viable cell when the two treatments were separated by incubation at 37 degrees C. For simultaneous treatments of heat and X-rays, or treatments immediately following each other, the transformation frequency was about the same as, or lower than, with X-rays alone. When transformation was calculated based on the cells at risk, hyperthermia treatment reduced the X-ray transformation frequency in all cases. Thus hyperthermia may not pose a carcinogenic risk in cancer therapy and could decrease the oncogenic potential of X-rays, depending on the treatment sequence.

Animals↗
PubMed1985

Switch-on of transcriptase function in reovirus: analysis of polypeptide changes using 2-D gels.

Two-dimensional gel electrophoresis (IEF and SDS-PAGE) was used to examine virion polypeptide changes associated with switch-on of transcriptase function in reovirus. Results reveal that switch-on is correlated with altered electrophoretic behavior of a specific minor polypeptide (delta 1) which is present in intermediate subviral particles. A second finding is that each of the molecular weight classes of viral polypeptides exists as a series of subspecies with different isoelectric points. This suggests that extensive posttranslational modification of progeny viral polypeptides occurs during particle morphogenesis. These findings have important theoretical and practical implications.

DNA-Directed RNA Polymerases↗
PubMed1985

In vitro transformation by bromodeoxyuridine and X irradiation in C3H 10T1/2 cells.

The effect of 10(-5) M bromodeoxyuridine (BrdUrd) substitution in C3H 10T1/2 cells was evaluated. Cellular toxicity increased rapidly for BrdUrd exposure times that were longer than the population doubling time. Radiosensitization by BrdUrd exposure was almost complete after one cell doubling time and was characterized by a decrease in D0 and the survival curve shoulder. Exposure to BrdUrd for one cell doubling time produced only very low transformation levels, but for prolonged BrdUrd exposure times, the transformation frequency per viable cell increased significantly. BrdUrd incorporation also enhanced radiation induction of transformation above the transformation levels resulting from the independent action of X rays or BrdUrd treatment. These results show that BrdUrd is a transforming agent in C3H 10T1/2 cells and thus may be a carcinogen and that BrdUrd can enhance radiation-induced transformation.

Animals↗
PubMed1985

Comparison of heat and/or radiation sensitivity and membrane composition of seven X-ray-transformed C3H 10T1/2 cell lines and normal C3H 10T1/2 cells.

C3H 10T1/2 mouse embryo cells were transformed by X-irradiation, and seven transformed clones were isolated and propagated as cell lines. Some of these cell lines produced tumors in syngeneic mice and grew in agarose while the normal C3H 10T1/2 cell line did not possess these characteristics. Exponentially growing cell cultures with comparable cell-cycle distributions as measured by flow cytometry were tested for heat and X-ray sensitivity. The heat and X-ray sensitivity varied randomly compared to the normal cell line. One cell line was more heat resistant and one more heat sensitive than the normal cell line, and the others had sensitivities comparable to the normal cell line. Measurements on some of the biochemical parameters of the particulate fraction of cells after sonication and 24,000 X g centrifugation showed that altered thermal sensitivity was not correlated with protein, cholesterol, or phospholipid content of this fraction.

Animals↗
PubMed1984

Effects of Ca2+ and Mg2+ on the switch-on of transcriptase function in reovirus in vitro.

Effects of Ca2+ and Mg2+ on the "switch-on" of transcriptase function in reovirus intermediate subviral particles were examined. Switch-on in this system is triggered by K+ ions, whereas Na+ ions antagonize the effects of K+ ions. Once effected, switch-on is irreversible. In the absence of Ca2+ and Mg2+ ions, switch-on of transcriptase is effected by incubation at 37 degrees C in the presence of a particular ratio of K+/Na+ (total K+ plus Na+ being kept constant). Inclusion of Ca2+ in the activation step lowers the ratio that triggers the switch-on event. This indicates that Ca2+ destabilizes the switched-off state of the transcriptase. Inclusions of Mg2+ ions in the activation step raises the K+/Na+ ratio necessary to trigger switch-on. Thus Mg2+ tends to stabilize the switched-off state of the transcriptase to the triggering action of K+ ions. Inclusion of both Ca2+ and Mg2+ together during the activation step results in mutual inhibition of the effects expected from each divalent cation alone. These results indicate that switch-on of transcriptase function, which is triggered by K+ ions, can be modulated by interactions involving the major physiological cations (Na+, K+, Mg2+, Ca2+).

Calcium↗
PubMed1984

Effects of oxygen and misonidazole on cell transformation and cell killing in C3H 10T1/2 cells by X rays in vitro.

The effects of oxygen (air) and misonidazole on the transformation and killing of 10T1/2 cells by X rays were examined. The oxygen effect for the cell transformation end point was very similar to that for cell killing. Misonidazole enhanced both cell killing and cell transformation to a similar extent. The enhancement of both end points by misonidazole occurred only in the absence of oxygen during irradiation and was of lesser magnitude than that observed for oxygen. These results demonstrate that the radiation chemical processes leading to cell killing and cell transformation, respectively, are affected similarly by these two enhancers of radiation action.

Aerobiosis↗
PubMed1984

Modification by anisotonic treatment of repair and fixation of radiation damage in cell killing and transformation.

Anisotonic treatment of V79 cells after irradiation resulted in extensive fixation of potentially lethal damage (PLD). Fixation of PLD occurred more rapidly when treated with 1.5 mol l-1 NaCl than with 0.05 mol l-1 NaCl. These effects were further enhanced when cells were held at 0 degree C instead of 37 degrees C during irradiation. When the X-ray (10.0 Gy) and salt treatments were separated by incubation at 37 degrees C, survival increased rapidly by a factor of 2000, while repair of PLD in cells held in a plateau phase after X-rays (10.0 Gy) caused survival to increase four-fold. The data indicate that these mammalian cells sustain and repair a much larger amount of PLD than is measured by the conventional method of holding cells in a non-progressing state after irradiation. Radiation sensitivity in cells was modified by BrdUrd incorporation into cellular DNA or exposure to DMSO during irradiation. For these cells, fixation of damage by post-irradiation anisotonic treatment proceeded at the same rate for BrdUrd-treated cells, and at a diminished rate for DMSO-treated cells, compared with cells irradiated in the absence of these compounds. In C3H-10T 1/2 cells, post-irradiation anisotonic treatment (with 0.05 or 1.5 mol l-1 NaCl) also resulted in fixation of PLD and a rapid recovery when the X-ray and salt treatments were separated by incubation at 37 degrees C. Treatment with 0.05 or 1.5 mol l-1 NaCl solutions immediately after irradiation also resulted in elevated transformation frequencies, which increased with exposure time to anisotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Perturbation of the switch-on of transcriptase activity in intermediate subviral particles from reovirus.

Intermediate subviral particles (ISVP) derived from reovirus represent a simple model system for the switch-on of transcriptase function. In such particles the endogenous transcriptase is present in a switched-off form, one step removed from the switched-on state. Switch-on of transcriptase function is an active process in this system and can be triggered by K+ions. A variety of agents which affect gene expression in cells were tested for an effect on switch-on in ISVP. Marked effects on switch-on in ISVP were observed with a diverse group of test agents, including DMSO and other solvents, BUdR, TdR, caffeine, theophylline, and temperature. The correlation in response between ISVP and cells suggests that the ISVP system may be useful as a model for studying the biochemical mechanisms underlying the perturbative effects of such agents on gene expression in cells.

Animals↗
PubMed1979

Two modes of entry of reovirus particles into L cells.

Evidence is presented supporting the hypothesis that reovirus intermediate subviral particles (ISVP), which show increased infectivity relative to intact virions, can gain entry into host L cells by two alternative pathways. One pathway is by the process of viropexis, involving phagocytic vacuoles. A second entry pathway is via direct penetration of the plasma membrane of the cell, without involvement of a phagocytic vacuole. Using electron microscopy, a kinetic analysis of the uptake process was carried out. Results indicate that at 37 degrees C ISVP gain entry into host cells primarily by direct entry, although viropexis also occurs, while intact virions gain entry by viropexis almost exclusively. A second line of experimental evidence consistent with the idea that ISVP can 'melt' their way through the plasma membrane is provided by studies on the release of pre-loaded radioactive 51Cr from host cells following infection. 51Cr release data demonstrate that infection with ISVP leads to an immediate increased leakiness of the cell plasma membrane, whereas no such increase takes place following infection with an equivalent number of intact virions. This demonstrates that ISVP can interact with the plasma membrane of the cell in a manner which is qualitatively different from the interaction between intact virions and the plasma membrane. The ability of ISVP to directly penetrate the plasma membrane of the host cell, which intact virions apparently cannot do, could explain the decreased duration of the eclipse phase, as well as the increased infectivity of ISVP, relative to that observed for infection with intact virions.

Adsorption↗
PubMed1979

Ionizing radiation perturbs the switch-on of transcriptase in a model transcription complex in vitro.

Ionizing radiation markedly alters the response of the reovirus transcriptase unblocking mechanism to stimulation by K+ ions, which normally trigger the switch-on of transcription in this system. In irradiated subviral particles the concentration of K+ ions needed to trigger switch-on is reduced in a dose-dependent way. The observed alteration of switch-on characteristics appears to correlate with alteration of the electrophoretic behaviour of a single major polypeptide species. These observations have important implications for understanding some of the effects of ionizing radiation on cells, most notably the induction of both latent virus and cell differentiation.

DNA-Directed RNA Polymerases↗