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Biomedical subjects

D M Cooper

Publications and source records attributed to D M Cooper.

At least 73 records · Page 4Linked to original sources
PubMed1998

Factors affecting ectopic gene conversion in mice.

Duplicated genes and repetitive sequences are distributed throughout the genomes of complex organisms. The homology between related sequences can promote nonallelic (ectopic) recombination, including gene conversion and reciprocal exchange. Resolution of these events can result in translocations, deletions, or other harmful rearrangements. In yeast, ectopic recombination between sequences on nonhomologous chromosomes occurs at high frequency. Because the mammalian genome is replete with duplicated sequences and repetitive elements, high levels of ectopic exchange would cause aneuploidy and genome instability. To understand the factors regulating ectopic recombination in mice, we evaluated the effects of homology length on gene conversion between unlinked sequences in the male germline. Previously, we found high levels of gene conversion between lacZ transgenes containing 2557 bp of homology. We report here that genetic background can play a major role in ectopic recombination; frequency of gene conversion was reduced by more than an order of magnitude by transferring the transgenes from a CF1 strain background to C57BL/6J. Additionally, conversion rates decreased as the homology length decreased. Sequences sharing 1214 bp of sequence identity underwent ectopic conversion less frequently than a pair sharing 2557 bp of identity, while 624 bp was insufficient to catalyze gene conversion at significant levels. These results suggest that the germline recombination machinery in mammals has evolved in a way that prevents high levels of ectopic recombination between smaller classes of repetitive sequences, such as the Alu family. Additionally, genomic location appeared to influence the availability of sequences for ectopic recombination.

Animals↗
PubMed1998

Ca2+-sensitive adenylyl cyclases, key integrators of cellular signalling.

The concept of second messenger signalling originated from the discovery of the role of cyclic AMP, although it is now known that cytosolic calcium [Ca2+]i mediates numerous signalling pathways and plays an equally vital role in many cellular events. In the last few years there has been a great deal of interest in the substantial molecular and functional diversity of mammalian adenylyl cyclases (ACs). Although AC was viewed as a generic activity, which was either stimulated or inhibited by stimulatory or inhibitory receptors, respectively, acting via alpha-subunits of trimeric GTP-regulatory proteins, the recent cloning of nine full-length isoforms, which significantly differ in their regulatory properties and tissue distributions, has revealed an unexpected level of complex regulation. In fact, each AC may integrate convergent inputs from many distinct signal-generating pathways. The nine isoforms can be divided into four distinct families, which reflect their distinct patterns of regulation by betagamma subunits of G-proteins, protein kinase C (PKC) and Ca2+. The mechanisms of regulation are often highly synergistic or conditional, suggesting a function of ACs as coincident detectors. Since all nine isoforms can be regulated either directly or indirectly by Ca2+ or PKC, a complex range of responses is possible. The Ca2+ concentration that stimulates the major ACs in brain has been found to inhibit AC activity in a number of peripheral tissues and cell lines. The purpose of this article is to review many of the important aspects about the distinct regulatory properties and cellular distribution of Ca2+-regulated ACs. Indeed, the notion that Ca2+ and cAMP are "synarchic" messengers acting in concert to regulate cellular activity was formally proposed some time ago. Here, we will focus on acute interactions between Ca2+ and cAMP and attempt to understand how AC activities can be regulated by discrete, physiological [Ca2+]i rises in intact cells. All Ca2+-regulated isoforms have characteristic distribution patterns in the brain. Also discussed are emerging insights on the temporal and spatial regulation of Ca2+- and cAMP-regulated pathways which may enable cell stimuli to elicit specific responses.

Adenylyl Cyclases↗
PubMed1998

Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog.

DMC1 is a meiosis-specific gene first discovered in yeast that encodes a protein with homology to RecA and may be component of recombination nodules. Yeast dmc1 mutants are defective in crossing over and synaptonemal complex (SC) formation, and arrest in late prophase of meiosis I. We have generated a null mutation in the Dmc1 gene in mice and show that homozygous mutant males and females are sterile with arrest of gametogenesis in the first meiotic prophase. Chromosomes in mutant spermatocytes fail to synapse, despite the formation of axial elements that are the precursor to the SC. The strong similarity of phenotypes in Dmc1-deficient mice and yeast suggests that meiotic mechanisms have been highly conserved through evolution.

Adenosine Triphosphatases↗
PubMed1998

Fitness and the effect of exercise training on the dietary intake of healthy adolescents.

OBJECTIVE: In healthy, nonobese, adolescent males and females to: (1) Determine the relationship between fitness and energy intake; (2) assess the effect of five-weeks endurance training on energy intake and food choice and (3) compare food record assessments of energy intake with doubly-labeled water (DLW) measurement of total energy expenditure (TEE). DESIGN: (1) Cross sectional analysis of fitness and food intake and (2) Prospective, randomized, controlled interventional study of endurance-type exercise training in 44 females and 44 males (age range, 15-17 y). MEASUREMENTS: Pre and end interventional three day food records were successfully collected from 32 females (15 controls, 17 trained) and 39 males (19 controls, 20 trained). Fitness was assessed from cycle ergometry as peak oxygen uptake normalized both to thigh muscle mass and body weight. Thigh muscle mass was measured by magnetic resonance imaging. TEE using the DLW technique was measured in 12 females (6 controls, 6 trained) and 20 males (10 controls, 10 trained) during weeks 4-5 of the exercise training program (simultaneously with the second assessment of food records). Food record data were analyzed using the Minnesota Nutrition Data System. RESULTS: Fitness was correlated with self reported total caloric intake in males but not females. In females, there was a significant increase in fat intake (19.8+/-9%, P < 0.05) and a significant decrease in carbohydrate intake (-9.8+/-4%) in the trained subjects. No changes were observed in the control subjects. Energy expenditure (2072+/-52 kcal/d) was significantly greater than the estimated energy intake (1520+/-112 kcal/d, P < 0.007) during the intervention in the trained, but not control, subjects. However, there was no weight change in either control or trained subjects. In males, no changes were observed in food choice in either control or trained subjects. Similar to the females, energy expenditure (2425+/-22 kcal/d) was significantly greater than the estimated energy intake (2168+/-117 kcal/d, P < 0.05) during the intervention in the trained, but not control, subjects. No weight changes were observed in either group. CONCLUSIONS: Fitness is associated with increased self-reported energy intake in males but not females, while exercise training led to alterations in food selection (greater fat and reduced carbohydrate) only in females. These observations could reflect specific gender differences, or, alternatively, the generally lower levels of fitness in the females. The apparent negative energy balance without evidence for weight loss in both the trained males and females suggests a systematic under reporting of food intake during exercise programs in adolescents, and indicates the possibility that errors in self reported food intake might be greater during transitions from one level of energy expenditure to another.

Adolescent↗
PubMed1998

Erythrocyte insulin-like growth factor-L binding in younger and older males.

OBJECTIVE: Insulin-like growth factor-1 (IGF-l) levels are lower in older compared with younger subjects. We tested the hypothesis that the reduction in circulating IGF-l would be accompanied by upregulation in tissue IGF-l binding in at least some tissues. We tested erythrocyte IGF-l binding since blood is an accessible tissue in humans, and there is growing evidence to suggest that erythrocyte IGF-l binding is influenced by circulating IGF-l. DESIGN AND PATIENTS: We compared 9 healthy older males (61-68 years old) with 9 healthy younger males (15-19 years old). MEASUREMENTS: Standard techniques were used to assay circulating IGF-l and IGF binding proteins 1-5 (IGFBPs 1-5). Erythrocyte IGF-l binding was first measured by studies in which native [125l]-IGF-l was displaced with unlabelled native IGF-l. In order to determine a possible role for IGF binding proteins (IGFBP), native [125l]-IGF-l was displaced with des-(1-3)IGF-1, which binds with IGF receptors but not IGFBPs. RESULTS: As expected, circulating IGF-l was significantly lower in the older compared with the younger subjects. In addition, IGFBP-3 and 5 were significantly lower, and IGFBP-4 higher, in older compared with younger subjects. When native [125l]-IGF-l was displaced with unlabelled native IGF-l, the number of IGF-l binding sites per erythrocyte was higher in the older subjects (43 +/- 5 vs. 18 +/- 2, older vs. younger, respectively; P < 0.05). In contrast, when native [125l]-IGF-l was displaced with des-(1-3), IGF-l binding capacity was not different between the two age groups. CONCLUSIONS: Erythrocyte IGF binding was increased in older compared with younger subjects. Surprisingly, the mechanism of the increase may not be a simple up regulation of IGF-l receptors in response to reduced circulating IGF-l, but possibly by an increase in the levels of as yet unidentified erythrocyte membrane-associated IGF binding proteins.

Adolescent↗
PubMed1998

Peak oxygen uptake, muscle volume, and the growth hormone-insulin-like growth factor-I axis in adolescent males.

PURPOSE: The growth effects of exercise appear to be mediated in part by central neuroendocrine control reflected in circulating levels of growth hormone (GH), insulin-like growth factor-I (IGF-I), and their binding proteins (BP). In previous studies positive correlations between peak VO2 and circulating IGF-I have been demonstrated. The relationship between peak oxygen uptake and these potential regulating factors has not been examined in adolescent males where patterns of GH pulsatility and levels of IGF-I are rapidly changing. METHODS: Forty-three healthy adolescent males (age 16 +/- 0.7 yr, 70% at Tanner V) performed cycle ergometry to determine p oxygen uptake (peak VO2), and magnetic resonance images to determine the thigh muscle volume. Baseline blood samples were collected for GHBP, the extracellular portion of the GH tissue receptor (by ligand mediated immunofunctional assay), IGF-I (by RIA), and IGFBPs 1-5 (by RIA). Mean GH was determined from samples obtained every 20 min overnight. RESULTS: Peak VO2/kg was positively correlated with mean overnight GH levels (r = 0.41, P < 0.005). Both peak VO2/kg and thigh muscle volume/kg were negatively correlated with GHBP (r = -0.33, P < 0.02) and IGFBP-4 (r = -0.52, P < 0.005). There were no correlations between peak VO2/kg and IGF-I or IGFBPs 1-3, and 5. CONCLUSIONS: GH pulsatility is increased adolescent males who have higher peak VO2, but this did not translate into increases in IGF-I. We speculate that in the fitter males, lower GHBP levels may reduce hepatic sensitivity to GH. Thus, circulating IGF-I was unchanged despite higher mean GH in subjects with higher peak VO2. IGFBP-4 which is known to inhibit IGF-I was negatively correlated with peak VO2 leading, possibly, to increased IGF-I bioactivity. Fitness (as assessed by muscle mass and peak VO2) does modulate the GH-IGF-I axis, but not solely through circulating IGF-I; both GHBP and IGFBPs play important roles.

Adolescent↗
PubMed1998

Increased physical activity and the growth hormone-IGF-I axis in adolescent males.

Insulin-like growth factor-I (IGF-I) is associated with muscle hypertrophy, and circulating IGF-I levels are correlated with fitness. To test the hypothesis that IGF-I increases with increased physical activity in adolescent males, 38 subjects (16 +/- 0.7 yr old) were randomized to control (n = 18) or increased physical activity groups for 5 wk. Before and after the intervention, we measured thigh muscle volume using magnetic resonance imaging and serum levels of mean growth hormone (GH) by overnight multiple sampling, GH binding protein (GHBP), IGF-I, and IGFBPs 1-5 by standard assays. Energy expenditure was assessed with the doubly labeled water technique toward the end of the study. In the training subjects there was 1) a significant increase in thigh muscle volume (+3.6 +/- 1%), 2) 15.5 +/- 3.3% greater energy expenditure than in controls, and 3) no evidence of weight loss (+1.44 +/- 0.4%). In contrast to our hypothesis, but similar to our recent observations in adolescent females, training decreased IGF-I (-12 +/- 4%, P < 0. 005). Moreover, training substantially reduced GHBP (-21 +/- 4%, P < 0.00002) and increased IGFBP-2 (+40 +/- 16%, P < 0.008). Brief training increased muscle volume in weight-stable adolescent males and, surprisingly, influenced not only IGF-I but GHBP and IGFBP-2 as well in a manner typically found in energy-deficient states.

Adolescent↗
PubMed1997

Construction of a full-length Ca2+-sensitive adenylyl cyclase/aequorin chimera.

Ca2+-sensitive adenylyl cyclases are key integrators of Ca2+ and cAMP signaling. To selectively probe dynamic changes in [Ca2+]i at the plasma membrane where adenylyl cyclases reside, a full-length, Ca2+-inhibitable type VI adenylyl cyclase/aequorin chimera has been constructed by a two-stage polymerase chain reaction method. The expressed adenylyl cyclase/aequorin chimera was appropriately localized to the plasma membrane, as judged by biochemical fractionation and functional analysis. The chimera retained full adenylyl cyclase activity and sensitivity to inhibition by physiological [Ca2+]i elevation. The aequorin portion of the chimeric construct was also capable of measuring changes in [Ca2+] both in vitro and in vivo. When the plasma membrane-tagged aequorin and cytosolic aequorin were compared in their measurement of [Ca2+]i, they showed contrasting sensitivities depending on whether the [Ca2+]i originated from internal stores or capacitative entry. This is the first full-length enzyme-aequorin chimera that retains the full biological properties of both aequorin and a Ca2+-sensitive adenylyl cyclase. This novel chimeric Ca2+ sensor provides the unique ability to directly report the dynamics of [Ca2+]i that regulates this Ca2+-sensitive enzyme under a variety of physiological conditions. Since this chimera is localized to the plasma membrane, it can also be used to assess local changes in [Ca2+]i at the plasma membrane as distinct from global changes in [Ca2+]i within the cytosol.

Adenylyl Cyclases↗
PubMed1997

Diagnosis of proliferative enteritis in frozen and formalin-fixed, paraffin-embedded tissues from a hamster, horse, deer and ostrich using a Lawsonia intracellularis-specific multiplex PCR assay.

Proliferative enteritis (PE) is an enteric disease that has been reported in a variety of animals. It is caused by an obligate intracellular bacterium identified in swine as Lawsonia intracellularis. The organism can be detected ante-mortem in swine with PE using molecular diagnostic methods. The disease can be diagnosed post-mortem in all species by gross examination of tissues and special histologic staining procedures. In this study we extracted total DNA from frozen or formalin-fixed, paraffin-embedded tissues from cases of pig, hamster, horse, deer and ostrich PE. The samples were subjected to a multiplex PCR reaction using primers specific for a swine isolate of L. intracellularis. Identical sized PCR products were detected in samples from all animals with PE and the specificity of the PCR reaction for L. intracellularis was demonstrated by Southern-blotting and hybridization using specific probes. These results suggest that the intracellular organism of PE in these species are all very closely related to the causative agent of PE in swine, L. intracellularis. In addition, this multiplex PCR assay can be used to detect the organism in frozen or archival tissues, facilitating retrospective diagnosis of PE.

Animals↗
PubMed1997

Fitness, fatness, and the effect of training assessed by magnetic resonance imaging and skinfold-thickness measurements in healthy adolescent females.

The relation between fitness and adiposity is particularly relevant to adolescent females in whom fitness is known to decrease and fatness to increase. However, little is known about the interaction of these variables in normally active, nonobese subjects. Our major hypotheses were that adiposity would be inversely correlated with physical fitness and that even a relatively brief intervention would lead to measurable, site-specific changes in body fat. We used a cross-sectional protocol to correlate body adiposity with indexes of fitness and a prospective study design to examine body adiposity before and after a 5-wk period of endurance training in 44 nonobese females aged 15-17 y (control group, n = 22; training group, n = 22). Adiposity was assessed by magnetic resonance imaging of the abdomen and thigh as well as by standard skinfold-thickness measuring techniques. Fitness was assessed by using cycle ergometer measurements of maximal oxygen uptake (VO2max). There were significant negative correlations between VO2max normalized to body weight and subcutaneous abdominal, thigh, and skinfold estimates of fat. However, when VO2 max was normalized to muscle volume these correlations were not significant. Abdominal fat increased in direct proportion to body weight (scaling factor = 1.14 +/- 0.16) but thigh fat increased proportionately less (scaling factor = 0.38 +/- 0.12, P < 0.05). Training increased thigh muscle mass significantly only in the midmuscle region and prevented the observed fat increase in the distal thigh of the control subjects. Body fat distribution in adolescent females appeared to be affected by many factors, including overall body weight and the level of physical activity.

Adipose Tissue↗
PubMed1997

Comparison of the 16S ribosomal DNA sequences from the intracellular agents of proliferative enteritis in a hamster, deer, and ostrich with the sequence of a porcine isolate of Lawsonia intracellularis.

Proliferative enteritis is an enteric disease that affects a variety of animals. The causative agent in swine has been determined to be an obligate intracellular bacterium, Lawsonia intracellularis, related to the sulfate-reducing bacterium Desulfovibrio desulfuricans. The intracellular agents found in the lesions of different animal species are antigenically similar. In addition, strains from the pig, ferret, and hamster have been shown to be genetically similar. In this study we performed a partial 16S ribosomal DNA sequence analysis on the intracellular agent of proliferative enteritis from a hamster, a deer, and an ostrich and compared these sequences to that of the porcine L. intracellularis isolate. Results of this study indicate that the intracellular agents from these species with proliferative enteritis have high sequence similarity, indicating that they are all in the genus Lawsonia and that they may also be the same species, L. intracellularis.

Animals↗
PubMed1997

Hydroxychloroquine therapy of diffuse pulmonary sarcoidosis in two Australian male children.

Sarcoidosis is a chronic granulomatous disease, seen infrequently in children, with the capacity to affect multiple systems. The incidence and prevalence of the disease in children is largely unknown but is dependent on many factors including: age, race and geographical location. There have been very few studies of sarcoidosis in Australian (Aboriginal or white) children. Diffuse pulmonary sarcoidosis is very rare in non-Scandinavian white Caucasian children, yet in the winter of 1992 two Caucasian males, aged 11.1 and 10.5 years were seen at the John Hunter Hospital in Newcastle. Both boys had pulmonary sarcoidosis and after an initial trial of treatment with steroids responded well to hydroxychloroquine therapy.

Antimalarials↗
PubMed1997

Increase in muscle IGF-I protein but not IGF-I mRNA after 5 days of endurance training in young rats.

Five days of treadmill training in rats leads to increased muscle size and running time. This was used to examine the effect of exercise on circulating insulin-like growth factor I [IGF-I; radioimmunoassay (RIA)], local muscle (hindlimb) IGF-I (by RIA), and muscle IGF-I mRNA (by ribonuclease protection assay). Eight-week-old female Sprague-Dawley rats were divided into three groups: control (n = 10); single-exercise test (n = 10), untrained but with one maximal exercise test at the end of the study; and training (n = 16), trained for 5 days and one maximal exercise test on day 6. There were no differences among the groups with respect to circulating IGF-I. Muscle IGF-I protein in trained rats (4.2 +/- 1.5 ng/g of muscle tissue) was significantly greater than both control (0.27 +/- 0.1 ng/g) and single-exercise test (0.62 +/- 0.19 ng/g, P < 0.05 by analysis of variance). There was no difference among the groups in IGF-I mRNA gene expression. These data suggest that there is an early, marked, local muscle increase in IGF-I protein in response to exercise. This increase, however, may not be related to increased muscle IGF-I gene expression. Moreover, the IGF-I response was probably local in nature since it was not matched by any increase in circulating IGF-I.

Animals↗
PubMed1997

Exercise-induced changes in circulating growth factors with cyclic variation in plasma estradiol in women.

The effect of 10 min of high-intensity cycling exercise on circulating growth hormone (GH), insulin-like growth factors I and II (IGF-I and -II), and insulin-like growth factor binding protein 3 (IGF BP-3) was studied in nine eumenorrheic women (age 19-48 yr) at two different phases of the menstrual cycle. Tests were performed on separate mornings corresponding to the follicular phase and to the periovulatory phase of the menstrual cycle, during which plasma levels of endogenous estradiol (E2) were relatively low (272 +/- 59 pmol/l) and high (1,112 +/- 407 pmol/l), respectively. GH increased significantly in response to exercise under both E2 conditions. Plasma GH before exercise (2.73 +/- 2.48 vs. 1.71 +/- 2.09 micrograms/l) and total GH over 10 min of exercise and 1-h recovery (324 +/- 199 vs. 197 +/- 163 ng) were both significantly greater for periovulatory phase than for follicular phase studies. IGF-I, but not IGF-II, increased acutely after exercise. IGF BP-3, assayed by radioimmunoassay, was not significantly different at preexercise, and exercise, or at 30-min recovery time points and was not different between the two study days. When assayed by Western blot, however, there was a significant increase in IGF BP-3 30 min after exercise for the periovulatory study. These findings indicate that the modulation of GH secretion associated with menstrual cycle variations in circulating E2 affects GH measured after exercise, at least in part, by an increase in baseline levels. The acute increase in IGF-I induced by exercise appears to be independent of the GH response and is not affected by menstrual cycle timing.

Adult↗
PubMed1997

Evidence for increased bone formation following a brief endurance-type training intervention in adolescent males.

The effect of exercise training, particularly relatively brief periods, on bone turnover markers in adolescents has been poorly studied. Thirty-eight healthy males (16+/-0.7 years) participated in a 5-week summer school program in which 20 subjects were randomly assigned to a training group consisting of 2 h/day, 5 days/week of endurance exercise, and 18 subjects were assigned to a control group. Bone formation was assessed by measurements of circulating osteocalcin, bone-specific alkaline phosphatase (BSAP), and the C-terminal procollagen peptide (PICP). Bone resorption was assessed by urinary levels of free deoxypyridinoline cross-links (dPYR) and the C-(CTX) and N-terminal (NTX) telopeptide cross-links. Prior to training, there was a weak positive correlation between fitness and PICP (r = 0.27, p < 0.05), but no correlations were observed between fitness and either the other markers of bone formation or bone resorption. Training led to a significant increase in (1) osteocalcin (15+/-4%, p < 0.03), (2) BSAP (21+/-6%, p < 0.02), and (3) PICP (30+/-11%, p < 0.03) and to a significant decrease in NTX (-21 +/- 3%, p < 0.05). These bone turnover markers did not change in the control subjects (osteocalcin, 0+/-4%; BSAP, 2+/-4%; PICP, -4 +/- 6%; NTX, -6 +/- 4%). There was no change in urinary dPYR and CTX in either control or trained subjects. Fitness is only weakly, if at all, correlated with bone formation, but a relatively brief period of endurance training leads to a substantial increase in bone formation markers in adolescent males. School-based, short-term exercise training programs could play a role in enhancing bone formation in adolescents.

Adolescent↗
PubMed1996

Functional co-localization of transfected Ca(2+)-stimulable adenylyl cyclases with capacitative Ca2+ entry sites.

Three adenylyl cyclases (ACI, ACIII, and ACVIII) have been described, which are putatively Ca(2+)-stimulable, based on in vitro assays. However, it is not clear that these enzymes can be regulated by physiological rises in [Ca2+]i when expressed in intact cells. Furthermore, it is not known whether transfected adenylyl cyclases might display the strict requirement for capacitative Ca2+ entry that is shown by the Ca(2+)-inhibitable ACVI, which is indigenous to C6-2B glioma cells (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155). In the present study, ACI, ACIII, and ACVIII were heterologously expressed in HEK 293 cells, and conditions were devised that distinguished capacitative Ca2+ entry from both internal release and nonspecific elevation in [Ca2+]i around the plasma membrane. Remarkably, not only were ACI and ACVIII largely insensitive to Ca2+ release from stores, but they were robustly stimulated only by capacitative Ca2+ entry and not al all by a substantial increase in [Ca2+]i at the plasma membrane elicited by ionophore. (ACIII, reflecting its feeble in vitro sensitivity to Ca2+, was unaffected by any [Ca2+]i rise.) These results suggest a quite unsuspected, essential association of Ca(2+)-sensitive adenylyl cyclases with capacitative Ca2+ entry sites, even when expressed heterologously.

Adenylyl Cyclases↗