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Biomedical subjects

D E Harrison

Publications and source records attributed to D E Harrison.

At least 91 records · Page 5Linked to original sources
PubMed1988

Biomarkers of aging: tissue markers. Future research needs, strategies, directions and priorities.

Objective tests that allow early detection of deleterious changes with age are necessary to develop treatments enhancing the health span--the length of healthy life. Here we report tests of eight biological systems that can be performed in mice with no harm to the subjects. Male and female B6, CBA and F1 mice were used. While most test results correlated with chronological age in most genotypes, none predicted subsequent longevities in more than two genotypes. Surprisingly, the open field activity test that most consistently predicted longevities, did not correlate with chronological age. Six tests predicted beneficial effects of food restriction in F1 males, but only one correctly predicted the deleterious effects of the same food restriction regimen in B6 males. These results suggest that different biological systems age at different rates, that rates are affected by genotype and that an anti-aging treatment beneficial in one genotype may be harmful in another.

Aging↗
PubMed1988

Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells.

We estimated numbers of transplantable primitive stem cells (PSCs) and found evidence that the same PSC continuously produced circulating erythrocytes and lymphocytes. These estimations used the binomial formula on data from recipients of identical portions of marrow mixtures containing two distinguishable cell types. Analysis of variance was used to compare repeated tests within each recipient. Values of pi s or pi c, probabilities that two independently sampled cells were descended from the same PSC, were also estimated, as this does not require the unverified condition that all PSCs contribute equally to the differentiated cell population. Several months after transplantation, erythrocytes were descended from only a single PSC per 1-2 X 10(5) marrow cells injected, several times rarer than previously reported. Percentages of erythrocyte and lymphocyte types in each recipient were closely correlated, with r values ranging from 0.86 to 0.94, in groups receiving 2-8 X 10(5) marrow cells; apparently the same precursors repopulated both myeloid and lymphoid lines in each recipient, as expected of true PSCs. Our data did not fit the clonal succession model, which predicts sequential activation of new PSCs and deactivation of old. Between 76 and 154 days, differentiated erythrocyte precursors were probably exhausted, with no evidence for new precursor activation or for further change between 154 and 250 days. The percentage of newly produced erythrocytes (reticulocytes) of each donor type varied little when individual recipients were followed between 165 and 295 days after transplantation, and variances within recipients were similar at marrow doses from 8 to 200 X 10(5) cells, further contradicting models of sequential activation and deactivation of PSC clones. Thus, transplanted PSCs were continually active during much of the recipient's lifespan.

Analysis of Variance↗
PubMed1988

Aging and glucose homeostasis in C57BL/6J male mice.

Age-dependent changes in glucose homeostasis were assessed in specific pathogen-free C57BL/6J male mice. Increased islet size and pancreatic insulin content in old (21-25-month-old) mice were associated with lower nonfasting plasma glucose levels and improved clearance of either an oral or an i.p. administered glucose load in comparison with young, mature (4-5-month-old) males. The almost twofold increase in islet size correlated with a twofold increase of glucose-stimulated insulin secretion from perifused islets from 25-month-old males compared with 5-month-old males. These aging male mice did not become obese, and there were no fibrotic changes associated with the hyperplastic islets observed in the old males. Thus, the findings that glucose tolerance did not deteriorate with age, coupled with the lack of evidence for impaired beta cell responsiveness to glucose in old males, suggest that deterioration in glucose homeostasis is not an inevitable consequence of aging in the mouse.

Aging↗
PubMed1988

Properties of single potassium channels modulated by glucose in rat pancreatic beta-cells.

1. The patch clamp method has been used to examine the effect of glucose on single K+ channel currents recorded from cell-attached patches on dissociated rat pancreatic beta-cells. Patch pipettes contained a 140 mM-K+ solution. 2. In glucose-free solution three types of K+ channels were observed. Two of these, having conductances of around 50 pS (G-channel) and 20 pS when the external K+ concentration, [K+]0, was 140 mM, were active at the resting potential of the cell. The G-channel was observed in more patches and showed higher activity; it therefore appears to contribute the major fraction of the resting K+ permeability of the beta-cell. At membrane potentials positive to about +20 mV a third type of K+ channel, having a mean conductance of 120 pS, was activated. The open probability of this channel was strongly voltage dependent and increased with depolarization. 3. The reversal potential of the G-channel current was shifted 59 mV by a 10-fold change in external K+ (Na+ substitution) indicating the channel is highly K+ selective. The single-channel conductance varied with [K+]o as predicted from the Goldman-Hodgkin-Katz equation; at physiological [K+]o (5 mM-K+) an inward conductance of around 10 pS is predicted. The amplitude of the single-channel current showed a tendency to saturate with increasing [K+]o. 4. Single G-channel currents show burst kinetics indicating at least two closed states. The open and closed (gap) times within the bursts were distributed exponentially with time constants of 2.5 ms (tau o) and 0.5 ms (tau c1) respectively at the resting potential of the cell. There was little change in tau c1 over the voltage range -40 to 60 mV (pipette potential) but tau o increased slightly with membrane depolarization. 5. The addition of glucose to the bath solution produced a reversible, dose-dependent decrease in G-channel activity. This decrease results principally from a reduction in the frequency and duration of the bursts of openings with increasing glucose. In addition, the mean open time decreases. The short gaps during the bursts were little affected by glucose. 6. At glucose concentrations of .10 mM and above the decrease in G-channel activity is accompanied by an increase in the input resistance of the cell and by the initiation of action potentials. 7. It is concluded that glucose metabolism results in a reduction of G-channel open probability and thereby produces depolarization of the beta-cell.

Action Potentials↗
PubMed1988

The decrease in long-term marrow repopulating capacity seen after transplantation is not the result of irradiation-induced stromal injury.

Marrow cells from nonirradiated F1-W/Wv mice repopulated slightly less well than cells from lethally irradiated recipients. Therefore, avoiding irradiation of recipients did not improve the relative repopulating ability of their marrow cells. In other experiments, F1-W/Wv mice were transplanted by parabiosis with marrow of WBB6F1-+/+ (F1-+/+) mice, avoiding cellular handling and irradiation. Marrow cells transplanted to F1-W/Wv mice by this procedure demonstrated slightly better repopulating ability than did marrow cells transplanted by injection. However, they performed no better than those transplanted by parabiosis to irradiated F1-+/+ recipients. Significant impairment of stromal function after irradiation was not indicated. Apparently, stem cell damage caused by transplantation may have greater importance in causing loss of stem cell replicative potential than effects of irradiation-induced stromal injury.

Animals↗
PubMed1988

Effects of transplantation and age on immunohemopoietic cell growth in the splenic microenvironment.

Intact spleens from young adult and aged mice were transplanted into young recipients to compare effects of age and effects of spleen transplantation on hemopoietic and immune functions. Hemopoietic functions of histocompatible spleen transplants were assessed by partial cures of genetically anemic WBB6F1-Sl/Sld recipients, and immune functions were measured as numbers of anti-SRBC PFC(sheep red blood cell plaque-forming cells) and responses to the mitogen PHA (phytohemagglutinin). Spleens from WCB6F1 and WBB6F1 donors at least 28 months old partially corrected anemias in 10 of 28 Sl/Sld recipients, whereas spleens from 5- to 10-month-old donors performed significantly better, partially correcting anemias in 22 of 31 Sl/Sld recipients. B6D2F1 spleens were transplanted from either old or young donors in B6D2F1 recipients to test their ability to support immune-responsive cells. These spleen grafts were much smaller than recipient spleens and contained few anti-SRBC PFC. In contrast WCB6F1-+/+ spleens transplanted in Sl/Sld recipients were much larger, weighing more than the intact spleens of the recipients. Nevertheless when these spleens were from young donors, they contained only about 10% as many anti-SRBC PFC and PHA-responsive cells as did recipient spleens, whereas old donor spleens contained even fewer. Use of splenectomized Sl/Sld recipients did not alter these results. Apparently the effect of transplantation was much more important than age in reducing the spleens' abilities to support immune-responsive cells.

Aging↗
PubMed1987

A semiquantitative measure of immune responses against erythropoietic stem cell antigens.

A semiquantitative assay was developed and used to measure the effects of immune responses against 16 independent non-H-2 antigenic loci on erythropoietic stem cells. The assay compares repopulation in genetically anemic WBB6F1-W/Wv recipients that have normal immune responses, and in lethally irradiated WBB6F1 +/+ mice whose immune responses are suppressed by the irradiation. The differences in repopulating ability between these two types of recipients measure how immune responses affect erythropoietic stem cells. Stem cell repopulating abilities for the cells with antigens specified by the Thy-1, H-1, H-24, Ly-1, H-37, and H-17 loci were affected slightly, if at all. Repopulating abilities were moderately reduced by responses against antigens specified by H-15, 16, Ea-2, and Ly-2, 3 loci, and against the differences between the B6 and B10 genotypes, although marrow of these types cured W/Wv recipients. A surprising result occurred for the antigen specified by the H-8 locus, in which immune responses strongly reduced repopulating abilities, although this type of marrow cell cured W/Wv recipients. A comparison of these results with skin graft survival times suggests that the antigens specified by the H-17 and H-24 loci are strongly immunogenic on skin but not on marrow stem cells, while those specified by the H-12 and H-8 loci are strongly immunogenic on marrow stem cells but not on skin.

Anemia↗
PubMed1987

Genetic differences in effects of food restriction on aging in mice.

Lifelong food restriction to two-thirds of normal ad libitum consumption extended mean and maximum life spans more than 200 d in male B6CBAF1 hybrid mice, already a long-lived genotype. The following biological systems were improved by food restriction, with values for older mice being similar to those previously found for younger individuals: tight wire clinging, a measure of neuromuscular performance; open field movement, a measure of voluntary activity; tail tendon denaturation rate, a measure of collagen solubility; urine concentrating ability, a measure of renal function, and hair regrowth rate, a measure of the frequency of hair follicle cycling. However, wound healing was slower in food-restricted mice than in ad libitum-fed controls. The same food restriction treatment had entirely different effects on longevities of a different genotype, male B6 (C57BL/6J) mice, reducing mean and maximum life spans 265 and 27 d, respectively. This surprising deleterious effect was not predicted by tests of tight wire clinging, open field movement and tail tendon denaturation, but was predicted by hair regrowth rates, as these were lower in restricted B6 mice than in fed controls. In genetically obese (ob/ob) B6 mice, food restriction extended mean and maximum longevities 327 and 440 d, yet no biological systems tested performed better than those of food-restricted normal (+/+) mice whose life spans were reduced. Thus the food restriction regimen that increased longevities for individuals of two genotypes decreased them for individuals of a third genotype tested in the same set of experiments.

Aging↗
PubMed1987

Effects of 2-ketoisocaproate on insulin release and single potassium channel activity in dispersed rat pancreatic beta-cells.

1. The effects of the insulin secretagogue 2-ketoisocaproate, and of arginine, on insulin release, intracellular adenosine triphosphate (ATP) concentration and the activity of single K channels in cell-attached membrane patches have been studied in primary cultures of beta-cells from adult rat islets of Langerhans. 2. Insulin secretion was significantly increased by 2-ketoisocaproate (20 mM). The time course of this release was biphasic. Arginine (20 mM) did not stimulate insulin secretion. 3. In the absence of 2-ketoisocaproate (or arginine), two kinds of K channel were regularly observed in cell-attached membrane patches held at the cell resting potential: a channel of approximately 20 pS conductance and a channel of 50 pS conductance ([K]o = 140 mM, room temperature). 4. Addition of 2-ketoisocaproate (20 mM) to the bath suppressed the activity of the 50 pS channel and initiated action potentials. Arginine (20 mM) was without effect. 5. The intracellular concentration of ATP was increased significantly by 2-ketoisocaproate (20 mM) but not by arginine (20 mM). 6. It is suggested that, like glucose, 2-ketoisocaproate depolarizes the beta-cell and mediates insulin secretion by reducing the open probability of the 50 pS K channel. The results are also consistent with the idea that both secretagogues inhibit this channel by increasing the cytoplasmic concentration of ATP.

Action Potentials↗
PubMed1987

Large numbers of primitive stem cells are active simultaneously in aggregated embryo chimeric mice.

The possibility has been repeatedly raised that erythropoiesis results from clonal succession--the differentiation of one or a very small number of the most primitive stem cells that are sequentially activated to proliferate forming clones of differentiated cells and then eventually decline, to be replaced by new stem cell clones. We studied this possibility in chimeric mice made by combining embryos from two different strains so that they would have two distinct stem cell populations, each of which produces a different hemoglobin type (d and s). These were compared with F1 hybrids in which every stem cell produces both types. We measured the percentage of type d in seven to ten serial samples of circulating reticulocytes taken at three- to seven-day intervals and found that the variability in percent of this hemoglobin was only slightly higher in the chimeric mice than in F1 controls; SD ranged from 2.7% to 5.5% in the chimeric mice and from 3.4% to 3.9% in the controls. Using the binomial formula, the numbers of new clones formed during the reticulocyte life span, approximately three days, ranged from 33 to 118 in the individual chimeric mice. However, these numbers are underestimates because estimated numbers of clones depend inversely on variabilities, and the calculations did not exclude the contribution of experimental error to the overall variability. Total percentages of type d hemoglobin were also measured in seven to nine successive serial samples at 60- to 136-day intervals. These gave mean values similar to measures of newly synthesized hemoglobin in the same mice, but SD were larger, ranging from 5.3% to 8.4%. This reflects experimental error, both because of excess day-to-day variability found in this type of measurement and because there could not be fewer primitive stem cells activated to form clones of erythrocytes during the 45-day erythrocyte life span than during the three-day life span of reticulocytes. Since most and maybe all of the variation between successive samples in the same chimeric mouse appear to result from experimental error, many or even all of the primitive stem cells may simultaneously contribute to erythropoiesis.

Animals↗
PubMed1987

Erythropoietic repopulating ability of stem cells from long-term marrow culture.

Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU-S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.

Animals↗
PubMed1986

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans.

Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Insulin secretory responses of a clonal cell line of simian virus 40-transformed B cells.

We have evaluated the potential of the clonal insulin-secretory cell line HIT-T15 as a model system for investigating stimulus-secretion coupling in pancreatic B cells. In contrast to other cell lines, HIT cell insulin secretion was consistently stimulated 2- to 3-fold by D-glucose. The maximally effective concentration of glucose was 10 mmol/l; between 2 and 10 mmol/l glucose the increase in insulin release was paralleled by an increased rate of glucose oxidation. The main characteristics of glucose-stimulated insulin release by HIT cells were essentially similar to those of normal islets. Thus, the response was specific for metabolizable sugars (D-mannose and D-glyceraldehyde stimulated insulin release but L-glucose and D-galactose were ineffective); markedly dependent on extracellular Ca2+ concentration; potentiated by forskolin, glucagon, acetylcholine and 12-O-tetradecanoyl phorbol 13-acetate; inhibited by adrenaline or somatostatin; showed a biphasic pattern of release in perifusion experiments, with both phases being potentiated by forskolin. The secretory response of the HIT cells to amino acids was also similar to that of normal islets. Thus, L-leucine and its deamination product 2-ketoisocaproate were effective stimuli, whereas L-isoleucine and L-glutamine were ineffective. Insulin release from HIT cells could also be evoked by the sulphonylureas glibenclamide and tolbutamide and by an increase in concentration of extracellular K+ to 40 mmol/l. The content of cyclic AMP in HIT cells was increased modestly by glucose but not by an increase in extracellular K+. Forskolin elicited a 4-fold increase in cyclic AMP content. We conclude that HIT cells retain the essential features of the insulin secretory response of normal B cells and represent an important tool for further biochemical characterization of the secretory system.

Animals↗
PubMed1986

Reactive perforating collagenosis: light, ultrastructural and immunohistological studies.

Reactive perforating collagenosis is an uncommon disorder and few accounts refer to ultrastructural features. This report includes a study by light and transmission electron microscopy of serially sectioned biopsies from early lesions in two patients. Immunohistological investigations utilizing antibodies to basement membrane, laminin, collagen and cytokeratin were also done. Collagen and elastin were demonstrated within the centre of the lesions and there was a defect in the basal lamina at the base of the lesion. The collagen, cytokeratin and the basal lamina in the lesions were antigenically similar to those in the surrounding normal skin. These results are compared with previous findings and discussed in the light of the current views on the pathogenesis of this disorder.

Basement Membrane↗
PubMed1986

Structure-activity relationships of alloxan-like compounds derived from uric acid.

The diabetogenic activity of a range of alloxan-like compounds derived from uric acid has been investigated. The classes of derivatives were: 5-substituted-isouric acids; 4,5-disubstituted-4, 5-dihydrouric acids; 5-substituted-pseudouric acids; salts of dehydro-uramil hydrate; salts of dehydro-isouramil hydrate; alloxan derivatives. Compounds were tested by intravenous injection into rats and diabetogenic activity assessed by production of persistent hyperglycaemia and glycosuria. The only essential structural feature common to all active compounds was the presence of a quinonoid pyrimidine system or its hydrated equivalent. The presence of the five-membered ring of uric acid (or an opened form thereof) did not abolish and in some compounds enhanced diabetogenic activity.

Alloxan↗
PubMed1985

Properties of isolated human islets of Langerhans: insulin secretion, glucose oxidation and protein phosphorylation.

In the present study, human islets were isolated by collagenase digestion from the pancreases of three kidney donors. Maintainance of the islets in tissue culture enabled insulin release, glucose oxidation and Ca2+ -calmodulin-dependent protein phosphorylation to be determined using the same islets. Increasing glucose over a range 0-20 mmol/l resulted in a sigmoidal stimulation of insulin release (28.8 +/- 5.2 to 118.4 +/- 25.8 microU . islet-1 . h-1, n = 10; threshold less than 4 mmol/l). There was a marked correlation between the insulin secretory response of the islets to glucose and their rate of glucose oxidation (5.9 +/- 0.3 at glucose 2 mmol/l up to 25.8 +/- 1.8 pmol . islet-1 . h-1 at 20 mmol/l, r = 0.98). N-acetylglucosamine (20 mmol/l) failed to elicit a secretory response from the islets. Stimulation of insulin secretion by glucose was dependent upon the presence of extracellular Ca2+. Extracts of the islets contained a Ca2+ -calmodulin-dependent protein kinase which phosphorylated a 48-kdalton endogenous polypeptide. Myosin light-chain kinase activity was demonstrated in the presence of exogenous myosin light chains. This report demonstrates for the first time the sigmoidal nature of glucose-stimulated insulin release from isolated human islets, and its correlation with enhanced glucose oxidation. Furthermore, this is the first report of the presence of Ca2+ -dependent protein kinases in human islets.

Adult↗
PubMed1985

Delayed reduction in T cell precursor frequencies accompanies diet-induced lifespan extension.

Recent experimentation has suggested that a decline in the proportion of lymphocytes that can respond to antigenic stimulation may contribute to the loss in immune function with increasing age. If a diminution of precursor frequency is a fundamental element of immunosenescence, then one would expect that manipulations which extend lifespan would also lead to a retarded decline in the measured proportion of reactive cell. Food restriction to about two-thirds of normal intake, initiated at weaning, has long been known to extend the lifespan of mice and rats. We show here that food restriction from weaning causes old mice to retain high levels of T cell precursors in both the helper and cytotoxic lineages. This association of delayed precursor cell loss with extended lifespan supports the hypothesis that changes in T cell precursor frequency may be a key component of age-related immunodeficiency.

Aging↗