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Biomedical subjects

D E Harrison

Publications and source records attributed to D E Harrison.

At least 127 records · Page 7Linked to original sources
PubMed1981

F1 hybrid resistance: long-term systemic effects sensitive to irradiation and age.

In contrast to the usual rapid growth of transplanted syngeneic marrow cells in spleens of lethally irradiated recipients, the growth of parental marrow cells from certain inbred strains of mice is resisted by their F1 hybrids, other strains or both. The full complexity of this well known natural resistance is demonstrated here by using three inbred strains and their three F1 hybrids in all parent-hybrid combinations of donor and recipient. A similar resistance to parental marrow grafts is reported here in W-anemic F1 hybrid recipients that are cured and repopulated without irradiation. Rather than resistance to short-term growth in spleens, F1-hybrid resistance to permanent repopulation of the entire hemopoietic system is studied here. This manifestation of hybrid resistance is radiosensitive and declines in recipients over the age of 12 months. Long-term hemopoietic repopulation is measured quantitatively by injecting mixtures of two marrow-cell types with distinguishable hemoglobins into stem-cell-deficient recipients. A very high degree of resistance is detected against WB but not B6 parental marrow when mixed with WBB6F1 marrow and injected into WBB6F1 recipients. Most, but not all, of this resistance to permanent, systemic repopulation is abrogated by irradiation of the recipients; it is also abrogated after they reach the age of 15 months. Mouse models of long-term hybrid resistance studied in the entire hemopoietic system may be particularly relevant for marrow transplantation in man, where the objective is long-term systemic repopulation.

Age Factors↗
PubMed1980

Evidence for the participation of calmodulin in stimulus-secretion coupling in the pancreatic beta-cell.

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.

Animals↗
PubMed1980

Studies on the induction and turnover of citrate-oxidizing capacity in Klebsiella aerogenes using chemostat culture.

Steady-state chemostat cultures of Klebsiella aerogenes growing on a carbon-limited medium were challenged with pulse of carbon sources, and the resultant changes in dissolved oxygen tension were shown to reflect changes in respiration rate. These changes were used to study the kinetics of induction of citrate-oxidizing capacity, which is most probably limited by citrate permease, and the return to the preinduced state. Previously unexposed cells showed a lag phase, the duration of which decreased with increasing growth rate, with a minimum of 10 min, followed by an induction phase of linear increase of citrate oxidation rate which continued as long as citrate was present. The rate of increase in activity, which can be equated to the rate of induction of citrate permease, was independent of citrate concentration but increased with growth rate. Previously exposed cells showed no lag and some residual activity before further induction. The kinetics of return to the preinduced state were unusual in that activity was short-lived with a half-life of 16 to 23 min while the lag took over 8 h to re-establish. The rate of decay of activity decreased with increasing growth rate.

Citrates↗
PubMed1980

Competitive repopulation: a new assay for long-term stem cell functional capacity.

A new assay for the long-term functional capacity of hemopoietic stem cells is reported. Stem cell function in each donor of a particular genotype is assayed by mixing its marrow cells with a constant number of marrow cells from a donor with distinguishable hemoglobin and measuring the relative ability of each donor to populate stem-cell-depleted recipients. For example, cells from many different B6 donors may be assayed by mixing them with a constant number of WBB6F1 cells from a single pool and injecting them into irradiated WBB6F1 recipients. As the ratio of B6 to WBB6F1 marrow cells increases from 0:1 to 3:1 the percentage of B6 hemoglobin increases in a linear fashion. This is also found with WB and WBB6F1 or CBA and B6CBAF1 mixtures. Correlation coefficients between the percentage of hemoglobin of one donor type and the ratio of cells in the mixture of that type ranged from 0.78 to 0.98 in single experiments, and were 0.68 and 0.75 using data pooled from several experiments.

Animals↗
PubMed1979

Proliferative capacity of erythropoietic stem cell lines and aging: an overview.

The earliest bone marrow precursor cell types, often called stem CELLS, have a very large capacity for self renewal. This makes them a useful model system in which to test the hypothesis that normal somatic cells have a limited proliferative capacity. Marrow precursor cells differentiate and multiply to replenish the supply of various blood cell types that constantly turn over. Especially with erythrocyte production, this function is well difined and can be tested rigorously to determine whether a significant amount of the stem cell proliferative capacity is exhausted. Functional tests generally show that marrow stem cell lines are exhausted after three to six serial transplantations into successive recipients; the few exceptions are cases in which functioning by cells from the irradiated recipients has not been ruled out. Genetic markers unambiguously identifying marrow stem cell lines from the original donor are necessary for clear cut interpretations of transplantation experiments. No significant differences are found when comparing erythrocyte production by marrow stem cell lines from old and young adult donors. This suggests that little or none of the erythropoietic stem cell's proliferative capacity is exhausted by a lifespan of normal functioning.

Aging↗
PubMed1979

Mouse erythropoietic stem cell lines function normally 100 months: loss related to number of transplantations.

Marrow stem cell lines from old and young donors in parallel experiments were transplanted into genetically anemic W/WV recipients. These recipients were populated and their anemias were cured by stem cell lines from WCB6F1 or C57BL/6 dorons that had been repeatedly transplanted up to five times at annual intervals into successive W/WV recipients. Old marrow cell lines produced erythrocytes normally for as long as 2600 to 3000 days. However, after three to four serial transplantations many stem cell lines failed to cure at least two-thirds of the recipients, and all failed by transplantation six. This decline occurred in a similar pattern whether the original stem line donor was old or young. Two experiments suggested that the decline was caused by the transplantation procedure: (1) chromosomally marked donor cells from old and young donors permanently populated lymph nodes in lethally irradiated recipients after the first transplantation, but under the same conditions cell lines from the same donors transplanted a second time were substantially infiltrated by regenerating recipient cells; (2) the ability to compete with the same chromosomally marked cell line in populating irradiated recipients declined markedly in both old and young marrow stem cell lines that had been previoulsy transplanted.

Aging↗
PubMed1979

Processing by the thymus is not required for cells that cure and populate W/WV recipients.

Adult marrow, fetal liver or nu/nu mouse marrow from histocompatible donors was grafted into genetically anemic W/WV recipients, and all three types of grafts cured thymectomized as well as intact W-anemic recipients. With the latter two types of graft, the genetic anemia was cured by cells that could not have been processed in a mature thymus, since the adult recipients were thymectomized before receiving the grafts, the nu/nu donors were congenitally thymusless, and the fetal donors were used at 16 days of gestation. Chromosome-marked marrow grafts were used to show that immune systems were populated to similar degrees in thymectomized and intact W/WV recipients. Therefore, the cells derived from the donor marrow graft that partially populate the immune systems of W-anemic recipients do not require thymus processing. Small numbers of liver rudiment or yolk sac cells from fetal donors less than 12 days old failed to cure W/WV recipients, even when mixed with adult thymus cells. Therefore, the lack of adequately developed thymic helper cells appears not to be the reason why early fetal hemopoietic stem cells fail to cure W/WV recipients.

Aging↗
PubMed1978

Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging.

Marrow stem cell lines from old donors and those from young controls gave equally rapid rates of colony growth on spleens of irradiated mice. Old and young stem cell lines competed equally well with chromosomally marked marrow stem cells from a young donor in producing cell types that are stimulated by bleeding; old cells competed 70% as well as young in producing cell types stimulated by phytohemagglutinin (PHA) in vitro. After a single serial transplantation, the rates of colony growth declined 1.5- to 2.5-fold, and the ability to compete declined 2- to 4-fold for bleeding-stimulated and 4- to 10-fold for PHA-stimulated cells. Thus, immediate stem cell proliferative capacities decline much more after one serial transplantation than after a lifetime of normal function.

Animals↗
PubMed1977

Molar growth yields, respiration and cytochrome profiles of Beneckea natriegens when grown under carbon limitation in a chemostat.

The effect of growth rate on the physiology of Beneckea natriegens was studied in chemostat culture. The molar growth yields (Y) from glucose and oxygen, the specific rates of oxygen (Qo2) and glucose (Qg1c) consumption and the specific rate of CO2 production (Qco2) were linearly dependent on the growth rate over the dilution rate 0.17 h-1 to 0.60 h-1. Further increase in the dilution rate resulted in a decrease in growth yield and respiration rate and these changes were coincident with increases in the specific rate of glucose utilisation and of acetate production. The affinity of Beneckea natriegens for glucose was similar when measured either directly in chemostat culture or in a closed oxygen electrode system using harvested bacteria. The total content of cytochromes decreased with increasing growth rate. However, the quantity of CO-binding cytochromes remained independent of growth rate and correlated with the potential respiration rate.

Carbon↗
PubMed1977

Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b.

1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed.

Ascorbate Oxidase↗
PubMed1977

Cell lines from old immunodeficient donors give normal responses in young recipients.

Two different immune responses were compared in spleen cells obtained from old and young CBA/HT6J mice. Spleen cells from old mice (23 to 33 months) responded about half as well as did spleen cells from young mice (4 to 10 months) in the adoptive transfer anti-sheep red blood cell (SRBC) plague-forming assay, and caused slightly less than half the uptake of tritiated thymidine in response to phytohemagglutinin (PHA) in vitro. Marrow stem cell from some of the old and young mice whose splenic immune responses were tested were transplanted into irradiated young CBA/CaJ recipients. Seven to 17 weeks later these same immune responses were tested in the spleen cells of these young recipients, and the T6 chromosome marker was used to identify donor cells. Old animals' responses varied greatly, perhaps due to suppressing cells or factors in some individuals. Therefore, cells were never pooled and the responses of receipients were compared to the responses of the donor whose marrow had populated them. The response for a particular old donor, or for the recipients of its stem cells, was divided by the response for the young control used with that donor, or for its stem cell recipients. This was called the old/young ratio. With original donors with an old/young ratio for the SRBC response of (mean +/- S.D.) 0.35 +/- 0.14, The old/young ratio for that same response in the recipients was significantly improved to 1.26 +/- 0.71. In original donors with an old/young ratio for the PHA response of 0.44 +/- 0.17, the old/young ratio in the recipients improved significantly to 0.86 +/- 0.27. Thus, little or none of the decline with age in these immune responses was intrinsic to the old lymphoid stem cells.

Aging↗