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N J Nelson

Publications and source records attributed to N J Nelson.

At least 73 records · Page 4Linked to original sources
PubMed1988

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.

Animals↗
PubMed1987

Characterization of radiolabeled fluoromisonidazole as a probe for hypoxic cells.

Radiolabeled fluoromisonidazole has been characterized as a probe for hypoxic cells in vitro and in vivo. The uptake and retention of [3H]fluoromisonidazole and [3H]misonidazole were compared in V-79 cell monolayers and spheroids by varying incubation time and O2 levels in contact with the medium. The two labeled drugs were retained similarly in cell populations isolated from different depths in spheroids, and the amount of each drug bound in cells at the spheroid periphery increased with decreasing O2 level. The labeling patterns in autoradiographs were similar for spheroids incubated with the two labeled drugs, with most silver grains located over a zone of viable and presumed hypoxic cells intermediate between the necrotic center and the periphery of the spheroid. Biodistribution of the two tritiated drugs was compared in C3H mice bearing KHT tumors with 15% radiobiologically hypoxic cells. Tumor:blood and tumor:muscle ratios greater than 5.0 were achieved in mice sacrificed 4 h after the last of three injections of 5 or 20 mumol/kg of [3H]fluoromisonidazole. These ratios are compatible with imaging and are higher than those obtained with 50 mumol/kg misonidazole in a similar administration protocol. TLC analysis of plasma from mice injected with [3H]fluoromisonidazole indicated that the drug was stable in vivo for up to 2 h and that the metabolites formed were too polar to be dehalogenation products. Fluoromisonidazole labeled with 18F at the end of the alkyl side chain would retain the label on metabolites that bind in hypoxic cells in vivo. Fluoromisonidazole binds stably in the same populations of hypoxic cells as does misonidazole, and we conclude that [18F]fluromisonidazole has potential use as a hypoxia imaging agent in vivo.

Animals↗
PubMed1987

Removal of transferrin from fetal bovine serum.

An antiserum against purified fetal bovine serum (FBS) transferrin was produced in rabbits. The isolation of anti-bovine transferrin IgG fraction was achieved by ammonium sulfate precipitation of rabbit hyperimmune plasma followed by ion exchange chromatography on diethylaminoethanol (DEAE) cellulose, at both an acidic and basic pH. The various antibody fractions were analyzed by fast protein liquid chromatography (FPLC) on a high-resolution mono-Q column. The specificity and efficiency of the antibody fractions obtained were tested by titration of a constant amount of fetal bovine serum with increasing amounts of antibody. The completeness of removal of the fetal bovine serum transferrin resulting from the formation of the highly stable antibody antigen complex was monitored by immunologic and radioisotope methods. The removal of FBS transferrin by this antibody precipitation technique did not interfere with the ability of added iron 59-tagged human transferrin to deliver iron to rat reticulocytes, as shown in an in vivo incubation model. The method proved to be effective for the fast and complete removal of bovine transferrin from fetal bovine serum in vitro and will be a prerequisite for more detailed studies of the interaction of transferrin of different species with tissue receptors on cell lines in culture without resorting to completely defined culture media.

Animals↗
PubMed1984

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution.

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.

Amifostine↗
PubMed1983

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo.

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.

Animals↗
PubMed1982

Tumor cell toxicity of stable gallium nitrate: enhancement by transferrin and protection by iron.

The cytotoxicity of citrated gallium nitrate (NSC 15200) to EMT-6/UW mouse sarcoma cells growing in vitro was assayed as growth inhibition in treated cultures as well as cell survival (colony-forming ability) after acute or chronic exposure to graded doses. Gallium nitrate is both cytostatic and lethal to cells, with some growth inhibition occurring after chronic exposure to low doses (10 micrograms/ml) which kill essentially no cells. Cell kill and growth inhibition were both observed if cells were exposed for 24 hr or more to doses greater than 50 micrograms/ml. The growth inhibitory and lethal effects of gallium nitrate were enhanced by the addition of human transferrin to the medium. This enhanced toxicity was consistent with, and proportional to, the increased gallium uptake in the presence of transferrin rather than a direct effect of this iron transport protein. The addition of ferric citrate greatly reduced the toxic effect of the gallium salt. Cells in stationary plateau phase cultures appear to be as sensitive to gallium nitrate as exponentially growing cells. Ga3+ may mimic Fe3+ in some aspects of cellular metabolism, and competition between the two metals occurs at the initial uptake step, binding to transferrin, and possibly at other points in cell metabolism.

Animals↗
PubMed1981

Increased frequency of the group 5a antigen in patients with hematologic malignancies.

We have confirmed our previously described finding that the group-5a antigen is associated with acute lymphoblastic leukemia. In addition we have found that the 5a antigen has an increased frequency in patients with chronic lymphocytic lymphoma but not in patients with acute myelogenous leukemia, acute myelomonocytic leukemia. Hodgkin's disease or histiocytic lymphoma.

Adult↗
PubMed1980

Effect of tumor dissaggregation on results of in vitro cell survival assay after in vivo treatment of the EMT-6 tumor: x-rays, cyclophosphamide, and bleomycin.

EMT-6 tumors were treated in vivo with 300 kVp X-rays, cyclophosphamide, or bleomycin. Tumor cell suspensions were prepared by digesting tumors with trypsin or a collagenase-deoxyribonuclease-pronase cocktail, and cells were plated in vitro for determination of fractional cell survival. Cell survival after X-rays was identical for the two disaggregation methods. Trypsin-derived cells were far more sensitive to bleomycin but less sensitive to cyclophosphamide than those prepared with the mixed enzyme cocktail. Interaction of drug produced and enzyme caused damage was the probable cause for these discrepancies. The nature of the interaction may be drug specific and therefore unpredictable. The results were unlikely to be due to different nonrepresentative tumor cell samples being produced by the two digestion methods, because the X-ray cell survival curves were so similar for the two products.

Animals↗
PubMed1980

Response of an in vivo-in vitro tumour to X-rays and cytotoxic drugs: effect of tumour disaggregation method on cell survival.

EMT-6/UW tumours were treated in vivo with X-rays, cyclophosphamide, or bleomycin. Cell survival was assayed in vitro following tumour disaggregation with trypsin or an enzyme cocktail (EC) consisting of pronase, collagenase and DNase which gives a 10-20 x higher cell yield. Surviving fraction was lower after cyclophosphamide treatment for cells isolated with EC than for cells prepared with trypsin. The opposite result was obtained with bleomycin; trypsin-isolated cells appeared more sensitive. In attempting to determine the basis for this discrepancy, it was found that both dissociation methods isolate a non-representative cell sample with fewer cells in DNA synthesis (12-13%) than in the original tumour (approximately 22%). The specific nature of the interaction between the injury caused by drug and enzyme remains to be elucidated.

Animals↗
PubMed1980

Common pathway for tumor cell uptake of gallium-67 and iron-59 via a transferrin receptor.

We studied the tumor uptake of [67Ga]citrate, [59Fe]citrate, and 125I-labeled transferrin (TF) by the in vitro growth form of EMT-6, a sarcoma-like mammary tumor of BALB/c mice. In analyzing the binding experiments, we developed a new mathematical model based on a formulation originally used to express the interaction of hormones with specific tissue receptors. The uptake of both carrier-free 67Ga and 59Fe by tumor cells was mediated by kinetically identical TF receptors. We also studied teric acid extracts of the stroma of EMT-6 tumors grown both in vivo and in vitro. Chromatography of these extracts on Sephacryl S-200 SF demonstrated that the cellular stroma contained specific TF-binding macromolecules. On the basis of these findings, we proposed the "transferrin receptor hypothesis" for the mechanism of 67Ga uptake by tumors. According to this view, a tumor-associated TF receptor is the functional unit responsible for the affinity of gallium for certain neoplasms. This receptor was also active in the uptake of iron by tumors.

Animals↗
PubMed1979

A transferrin-mediated uptake of gallium-67 by EMT-6 sarcoma. I. Studies in tissue culture.

We have studied the in vitro uptake of gallium-67 by exponentially growing EMT-6 sarcoma cells in long-term tissue culture. In this system, the addition of transferrin to the medium was required before an appreciable cellular uptake of Ga-67 occurred. The transferrin effect was complex, with an initial stimulation to a peak cell-to-medium ratio of 8--10:1 at low concentrations of transferrin (0.2 mg/ml), followed by a gradual decline in uptake as transferrin in the medium was increased further. EMT-6 tumor-cell uptake of Ga-67 was probably mediated by a specific cellular receptor for transferrin. Scatchard analysis of the EMT-6 cellular binding of human transferrin labeled with iodine-125 indicated a cellular receptor with affinity for transferrin of 5 X 10(6) l/mole and abundance of 500,000 receptors per cell. Over the experimental range of transferrin concentration in the medium, the observed uptake of Ga-67 was closely correlated with the degree of formation of Ga-67-labeled transferrin and the fraction of transferrin bound to the cellular receptor (N = 69, r = 0.86, p less than 0.0001).

Animals↗
PubMed1978

A stage-restricted secretory system in the submandibular gland of the neonatal rat.

Ribonuclease and amylase have been shown to undergo a transient increase in activity in the submandibular gland of the perinatal rat [5]. We report here that brief stimulation with isoproterenol in vitro selectively releases these enzymes. In addition, analysis by polyacrylamide gel electrophoresis showed that the 4-day old submandibular gland contains three other major protein species that are not present in the sublingual gland and two of which are not evident in the adult submandibular gland. These are also selectively released by the drug. Examination of the glands by light and electron microscopy showed that concurrent with release of these protein products, extensive degranulation occurs in the immature acini (terminal tubules). Our experiments suggest that the 4-day glands show a marked incorporation of 3H-leucine into the submandibular-specific, secreted protein species. This indicates that these proteins will provide convenient molecualr markers of early submandibular differentiation.

Amylases↗
PubMed1977

Cure of EMT-6 tumors by X rays or neutrons: effect of mixed-fractionation schemes.

EMT-6 mouse tumors were treated with (a) 1 or 5 fractions of either x rays or neutrons or (b) a mixture of both in which 2 fractions of neutrons (n) plus 3 fraction of x rays (x) were given in 5 days in the sequence n-n-x-x-x or n-x-x-x-n. Using local tumor control as an end point, neutron RBEs of 1.7 for single fractions and 2.6 for 5 fractions were obtained. The two mixed schemes yielded RBEs of 1.5 and 1.4, respectively. Therapeutic gain factors, calculated as the ratio of tumor to skin RBE with neutrons or mixed radiations, were highest for the mixed fractionation schemes. These results are due to an apparent enhancement of the neutron effect in the tumor but not in the skin with these regimens. Other normal tissues must be irradiated with mixed schemes to determine whether this phenomenon is limited to the skin.

Animals↗
PubMed1977

Effect of roentgen, cyclotron neutron, or mixed neutron-photon fractionated irradiation of mice. LD50/4 day values.

Mice were whole-body-irradiated with 5 fractions of roentgen rays in 5 days, 5 fractions of cyclotron neutrons in 5 days, or with mixed neutron-photon fractionated radiation, in the sequence n-n-x-x-x or n-x-x-x-n. The LD50/4 day values were determined. Roentgen rays and neutrons interact in the additive manner in the mixed fractionation schemes: effective dose per fraction is as predicted from the roentgen ray-only and neutron-only experiments. This essentially agrees with HENDRY et coll. (1976). However, no trend was found towards a less-than -additive effect which was observed by those authors and has also been suggested in skin response to mixed schemes (NELSON et coll. 1975).

Animals↗