Search PubMed⌕ Search

Biomedical subjects

M A Bender

Publications and source records attributed to M A Bender.

At least 55 records · Page 3Linked to original sources
PubMed1989

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

Animals↗
PubMed1989

Regulated expression of the human beta-globin gene after retroviral transfer into murine and human hematopoietic cells.

Retroviral vectors and infection protocols were developed which permit transfer in vitro of the human beta-globin gene into transformed erythroid cells and normal human and murine hematopoietic cells. In murine erythroleukemia (MEL) cells, RNA expression from the human beta-globin gene was regulated in parallel with the endogenous globin genes and this RNA directed synthesis of human beta-globin protein chains. Human BFU-E which were present in normal bone marrow samples were also infected with the globin virus. After erythroid maturation in vitro, several percent of the total beta-globin mRNA was derived from the virally transferred beta-globin gene in the erythroid progeny cells of the bursts. The initial design of the beta-globin vectors was improved after the removal of sequences which interfered with the production of high-titer retrovirus stocks. The improved vector can transfer the human beta-globin gene to pluripotential hematopoietic stem cells (PHSC) of the mouse as shown by long-term expression of human beta-globin RNA and protein in peripheral blood, and the presence of the globin provirus in reconstituted myeloid and lymphoid cell lineages in primary and secondary recipients of virus-infected bone marrow.

Animals↗
PubMed1988

Normal G2 chromosomal radiosensitivity and cell survival in the cancer family syndrome.

Recent reports have suggested that elevated chromosomal aberration yields following X-irradiation of skin fibroblasts in the G2 phase of the cell cycle are characteristic of affected members of cancer-prone families. These studies propose that this phenomenon is a consequence of impaired DNA repair and might be a useful predictor of genetic susceptibility to cancer. We have tested G2 chromosomal X-ray sensitivity in skin fibroblasts and peripheral blood lymphocytes from members of a kindred with the cancer family syndrome, a disorder in which susceptibility to colon cancer and other epithelial cancers is inherited in an autosomal dominant pattern. Further, using a cell survival assay, we tested cancer family syndrome skin fibroblasts for sensitivity to four classes of mutagens, including X-rays. In the assays used, skin fibroblasts and lymphocytes from both affected and unaffected family members exhibited responses indistinguishable from normal controls. Karyotypic analysis of lymphocytes and fibroblasts revealed no consistent constitutional cytogenetic abnormality. Thus, affected patients with the cancer family syndrome do not have increased sensitivity to irradiation and chemical mutagens and lack a germ-line chromosomal defect.

Cell Survival↗
PubMed1988

An investigation using inhibition of G2 repair of the molecular basis of lesions which result in chromosomal aberrations.

Cultures of JU56 cells were irradiated with 2.5 Gy X-rays and 16 h later the cultures were exposed to a moderately inhibitory dose of 1-beta-D-arabinofuranosylcytosine (ara-C) or aphidicolin (APC) and to colcemid, for 2 h. The c-metaphases collected for examination had therefore been exposed to X-rays in G1 or early S, and to the repair inhibitors APC and ara-C during the latter half of G2. It was found that treatment of cells irradiated early in cell cycle, that is, in G1 and early S, with APC or ara-C in G2, (1) reduced the frequency of chromatid and chromosome exchanges below that of cells treated with X-rays alone, (2) produced no more chromatid breaks and gaps than were seen in unirradiated cells, (3) increased the number of chromosome fragments and gaps in a more than additive fashion, and (4) produced only an additive effect, by comparison with the effect of X-rays and drug given separately, on the total number of chromosomal aberrations.

Animals↗
PubMed1988

Current status of cytogenetic procedures to detect and quantify previous exposures to radiation.

The estimation of the magnitude of a dose of ionizing radiation to which an individual has been exposed (or of the plausibility of an alleged exposure) from chromosomal aberration frequencies determined in peripheral blood lymphocyte cultures is a well-established methodology, having first been employed over 25 years ago. The cytogenetics working group has reviewed the accumulated data and the possible applicability of the technique to the determination of radiation doses to which American veterans might have been exposed as participants in nuclear weapons tests in the continental U.S.A. or the Pacific Atolls during the late 1940s and 1950s or as members of the Occupation Forces entering Hiroshima or Nagasaki shortly after the nuclear detonations there. The working group believes that with prompt peripheral blood sampling, external doses to individuals of the order of about 10 rad (or less if the exposure was to high-LET radiation) can accurately be detected and measured. It also believes that exposures of populations to doses of the order of maximum permissible occupational exposures can also be detected (but only in populations; not in an individual). Large exposures of populations can also be detected even several decades after their exposure, but only in the case of populations, and of large doses (of the order of 100 to several hundred rad). The working group does not believe that cytogenetic measurements can detect internal doses from fallout radionuclides in individuals unless these are very large. The working group has approached the problem of detection of small doses (less than or equal to 10 or so rad) sampled decades after the exposure of individuals by using a Bayesian statistical approach. Only a preliminary evaluation of this approach was possible, but it is clear that it could provide a formal statement of the likelihood that any given observation of a particular number of chromosomal aberrations in a sample of any particular number of lymphocytes actually indicates an exposure to any given dose of radiation. It is also clear that aberration frequencies (and consequently doses) would have to be quite high before much confidence could be given to either exposure or dose estimation by this method, given the approximately 3 decades of elapsed time between the exposures and any future blood sampling.(ABSTRACT TRUNCATED AT 400 WORDS)

Abnormalities, Radiation-Induced↗
PubMed1988

Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample.

In order to assess the potential of cytogenetic determinations on peripheral blood lymphocytes as a means of monitoring human populations subject to low level occupational and environmental exposures to chemical mutagens and carcinogens, accurate baseline data are required. Accordingly, we have determined mean frequencies of chromosomal aberrations and of sister-chromatid exchanges, their variances, and the sources of this variance in a cohort of 353 healthy employees of the Brookhaven National Laboratory. A detailed protocol was adopted for blood sampling, lymphocyte culture, cytogenetic preparation and scoring in order to minimize variation from these potential sources. Scoring was divided between the Oak Ridge and the Brookhaven groups with duplicate scoring sufficient to evaluate and minimize the effect of any differences between laboratories or between individual scorers. In all, the data include 71,950 cells scored for chromosomal aberrations and 16,898 cells scored for sister-chromatid exchanges. The mean unadjusted frequency of sister-chromatid exchanges was 8.29 +/- 0.08/cell. As reported in other studies, cigarette smoking very significantly influenced sister-chromatid exchange frequencies; in our study the mean for smokers was 9.0 +/- 0.2, while that for non-smokers was 8.1 +/- 0.1/cell. The mean frequency was statistically higher in females than in males, regardless of smoking status. On the other hand, age of the subject did not significantly influence sister-chromatid exchange frequencies. Curiously, the subject's total white cell count did influence sister-chromatid exchange frequency. No other source of variation was found. The frequencies of chromosomal aberrations of all types were determined. The frequency of the most common unequivocal chromatid type, the chromatid deletion, was 0.81 +/- 0.05%, that of the most common unequivocal chromosome type, the dicentric, was 0.16 +/- 0.02%. No statistically significant influence was found of age or sex, nor of any other parameter tested, on the frequency of any chromosomal aberration type, with the single exception of long acentric fragments, often "supernumerary", believed to represent X chromosomes precociously separated at the centromere. Such fragments were significantly more frequent in samples from females than those from males, and showed a significant positive regression on age.

Aneuploidy↗
PubMed1988

Chromosomal aberrations and sister-chromatid exchanges in lymphocytes from coke oven workers.

To test whether coke oven workers, an occupational group known to be at increased cancer risk, manifest increased peripheral blood chromosomal aberration frequencies, we obtained samples from a group of 30 steelworker volunteers, who had worked several years at coke oven jobs. Exposure estimates were made using measurements of work place atmospheric coal tar pitch volatiles and work histories. No statistically significant positive regression of chromosomal aberrations on exposure estimates was found. The data from the coke oven workers were also compared with the obtained concurrently and employing precisely the same laboratory protocol from a group of male Brookhaven National Laboratory employees. The coke oven workers as a group were found to have statistically significantly elevated frequencies of chromatid aberrations and of sister-chromatid exchanges.

Aneuploidy↗
PubMed1988

Design of retrovirus vectors for transfer and expression of the human beta-globin gene.

Regulated expression of the human beta-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective beta-globin genes. We found regions that interfered with virus production within intron 2 of the beta-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for beta-globin expression. Inclusion of beta-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, we found no effect of the antisense beta-globin transcription on virus production. A region downstream of the beta-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10(6) CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human beta-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human beta-globin gene in hematopoietic cells (CFU-S cells) in mice.

Animals↗
PubMed1988

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

Animals↗
PubMed1987

Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.

Gene Products, gag↗
PubMed1987

The synergistic effect of aphidicolin on the yield of X-ray-induced chromosome aberrations throughout the cell cycle in JU56 cells.

The effect of a 2-h post-treatment with aphidicolin at a dose sufficient to inhibit DNA synthesis on the yield of X-ray-induced chromosomal aberrations throughout the cell cycle was measured. Exposure to aphidicolin during and after irradiation brought about an increase in exchanges in cells irradiated in G2, in sister unions only in cells irradiated in S, and in all chromosome aberration types (fragments, sister unions, and dicentrics) in cells irradiated in G1. It is suggested that, during G1 and G2 but not during S inhibiting the repair enzyme alpha-polymerase brings about the conversion of some X-ray-induced DNA lesions to double-strand which can then take part in aberrations.

Animals↗
PubMed1986

Dose relationships between different effects of aphidicolin in JU56 cells.

Some effects of aphidicolin have been investigated in relationship to dose, in a permanent cell line, JU56. Inhibition of semi-conservative DNA synthesis occurred at concentrations greater than 3 X 10(-7) M. In this respect the cells were about as sensitive as L1210 and HeLa cells, and more than 10-fold more sensitive than PHA-stimulated human peripheral blood leucocytes. Delay of progress of cells through G2 occurred at concentrations which inhibited synthesis to about 2% of control levels. Chromatid aberrations appeared in cells at concentrations which decreased synthesis to 5%. Synergism with X-rays in the production of chromatid aberrations occurred at doses which reduced semi-conservative synthesis to 40% of control levels. Isochromatid aberrations appeared in cells continuously exposed to aphidicolin in G2 at concentrations which reduced synthesis to 5% of control units.

Animals↗
PubMed1986

Efficacy of dantrolene sodium in management of tetanus in children.

The effectiveness of dantrolene sodium in controlling tetanic spasms was evaluated in 21 children (aged 4 days-13 years) with tetanus. These dantrolene-treated patients were compared with an historical control group of 26 children previously treated for tetanus by traditional means at the same institution. The groups were comparable in terms of age, other treatments, and severity of disease. All patients received standard treatments for tetanus, including sedatives, antibiotics, and tetanus-immune globulin. Patients in the dantrolene-treated group also received dantrolene, either intravenously (Dantrium Intravenous) or orally, in divided doses totalling 4-6 mg/kg/day. Mortality was 73% (neonates 83%, non-neonates 50%) in the control group, compared with 33% (neonates 50%, non-neonates 0%) in the dantrolene-treated group. Dantrolene did not cause additional respiratory depression when administered alone or in combination with other drugs routinely used to treat tetanus. In this study, the use of dantrolene with traditional conservative therapy significantly reduced mortality from tetanus.

Adolescent↗