A longitudinal study of bone area, content, density, and strength development at the radius and tibia in children 4-12 years of age exposed to recreational gymnastics.

UNLABELLED: This study investigated the long-term relationship between the exposure to childhood recreational gymnastics and bone measures and bone strength parameters at the radius and tibia. It was observed that individuals exposed to recreational gymnastics had significantly greater total bone content and area at the distal radius. No differences were observed at the tibia. INTRODUCTION: This study investigated the relationship between exposure to early childhood recreational gymnastics with bone measures and bone strength development at the radius and tibia. METHODS: One hundred twenty seven children (59 male, 68 female) involved in either recreational gymnastics (gymnasts) or other recreational sports (non-gymnasts) between 4 and 6 years of age were recruited. Peripheral quantitative computed tomography (pQCT) scans of their distal and shaft sites of the forearm and leg were obtained over 3 years, covering the ages of 4-12 years at study completion. Multilevel random effects models were constructed to assess differences in the development of bone measures and bone strength measures between those exposed and not exposed to gymnastics while controlling for age, limb length, weight, physical activity, muscle area, sex, and hours of training. RESULTS: Once age, limb length, weight, muscle area, physical activity, sex, and hours of training effects were controlled, it was observed that individuals exposed to recreational gymnastics had significantly greater total bone area (18.0 ± 7.5 mm(2)) and total bone content (6.0 ± 3.0 mg/mm) at the distal radius (p < 0.05). This represents an 8-21 % benefit in ToA and 8-15 % benefit to ToC from 4 to 12 years of age. Exposure to recreational gymnastics had no significant effect on bone measures at the radius shaft or at the tibia (p > 0.05). CONCLUSIONS: Exposure to early life recreational gymnastics provides skeletal benefits to distal radius bone content and area. Thus, childhood recreational gymnastics exposure may be advantageous to bone development at the wrist.

Does lean tissue mass accrual during adolescence influence bone structural strength at the proximal femur in young adulthood?

UNLABELLED: The purpose of this study was to identify whether young adult bone structural strength at the hip is associated with adolescent lean tissue mass (LTM) accrual. It was observed that those individuals who accrued more LTM from adolescence to adulthood had significantly greater adult bone structural strength at the hip. INTRODUCTION: The purpose of this study was to identify whether young adult bone cross-sectional area (CSA), section modulus (Z), and outer diameter (OD) at the hip were associated with adolescent LTM accrual. METHODS: One hundred three young adult participants (55 males, 48 females) were tertiled into adolescent LTM accrual groupings. LTM accrual was assessed by serial measures using dual energy X-ray absorptiometry (DXA) from adolescence to young adulthood (21.3 ± 1.3 years). CSA, Z, and OD at the narrow neck (NN) and femoral shaft (S) sites of the proximal femur were assessed in young adulthood (21.3 ± 4.5 years), using hip structural analysis. Group differences were assessed using an analysis of covariance, controlling for adult height, weight, sex, and physical activity levels. RESULTS: It was found that individuals with higher adjusted adolescent LTM accrual had significantly greater adult adjusted values of NNCSA (2.49 ± 0.06 vs 2.77 ± 0.07 cm(2)), NN Z (1.18 ± 0.04 vs 1.37 ± 0.04 cm(3)), NN OD (3.07 ± 0.04 vs 3.21 ± 0.04 cm), SCSA (3.45 ± 0.08 vs 3.88 ± 0.09 cm(3)), and SZ (1.77 ± 0.05 vs 2.00 ± 0.05 cm(3)) than individuals with lower LTM accrual (p < 0.05). CONCLUSIONS: These findings suggest that the amount of LTM accrued from adolescence to young adulthood has a positive influence on adult bone structural strength at the proximal femur.

Tracking of aerobic fitness from adolescence to mid-adulthood.

BACKGROUND: Although adults' aerobic fitness is known to be correlated with cardiovascular disease risk, the longitudinal relationship with adolescent aerobic fitness is poorly described. AIM: To longitudinally investigate the relationship between aerobic fitness during adolescence and adulthood. SUBJECTS AND METHODS: Participants (207 boys, 149 girls) aged 7-17 years performed annual measures of VO2peak. In adulthood (40 and 50 years), 78 individuals (59 males and 18 females) were reassessed. Serial height measurements were used to estimate age at peak height velocity (APHV). During adolescence, VO2peak was measured via a treadmill test to voluntary exhaustion; adult VO2peak was assessed using submaximal predictive tests. Correlations were tested using Spearman's rho. ANCOVA was used to assess adult VO2peak group differences based off APHV VO2peak groupings (low, average or high). RESULTS: When sexes were pooled, moderate tracking existed from 2 years prior to APHV to APHV and APHV to 2 years after APHV (0.46, p < 0.001 and 0.35, p < 0.01, respectively). Correlations between APHV and adult values were low when sexes were pooled (p < 0.05). Comparisons of aggregated sexes revealed the low adolescent VO2peak group had lower values in adulthood relative to other groups (p < 0.05). CONCLUSION: Aerobic fitness has a low tracking between APHV and adulthood.

Macrophage growth arrest by cyclic AMP defines a distinct checkpoint in the mid-G1 stage of the cell cycle and overrides constitutive c-myc expression.

Proliferation of a murine macrophage cell line (BAC1.2F5) in response to colony-stimulating factor 1 (CSF-1) is inhibited by prostaglandin E2 (PGE2)-mediated elevation of intracellular cyclic AMP (cAMP). When BAC1.2F5 cells were growth arrested in early G1 by CSF-1 starvation and stimulated to synchronously enter the cell cycle by readdition of growth factor, PGE2 inhibited [3H]thymidine incorporation when added before mid-G1, but its addition at later times did not block the onset of S phase. Reversible cell cycle arrest mediated by a cAMP analog required the presence of CSF-1 for cells to initiate DNA synthesis, whereas cells released from an aphidicolin block at the G1/S boundary entered S phase in the absence of CSF-1. PGE2 or cAMP analogs did not block the initial induction of c-myc mRNA by CSF-1 but abolished the CSF-1-dependent expression of c-myc mRNA in the mid-G1 stage of the cell cycle. The cAMP-mediated reduction in c-myc RNA levels was due to decreased c-myc transcription. However, CSF-1-dependent BAC1.2F5 clones infected with a c-myc retrovirus were growth arrested by cAMP analogs despite constitutive c-myc expression. Therefore, the reduction of endogenous c-myc expression by cAMP is neither necessary nor sufficient for growth inhibition.

Phosphatidylcholine hydrolysis and c-myc expression are in collaborating mitogenic pathways activated by colony-stimulating factor 1.

Stimulation of diglyceride production via phospholipase C (PLC) hydrolysis of phosphatidylcholine was an early event in the mitogenic action of colony-stimulating factor 1 (CSF-1) in the murine macrophage cell line BAC1.2F5 and was followed by a second phase of diglyceride production that persisted throughout the G1 phase of the cell cycle. Addition of phosphatidylcholine-specific PLC (PC-PLC) from Bacillus cereus to the medium of quiescent cells raised the intracellular diglyceride concentration and stimulated [3H]thymidine incorporation, although PC-PLC did not support continuous proliferation. PC-PLC treatment did not induce tyrosine phosphorylation or turnover of the CSF-1 receptor. The major protein kinase C (PKC) isotype in BAC1.2F5 cells was PKC-delta. Diglyceride production from PC-PLC did not target PKC-delta, since unlike phorbol esters, PC-PLC treatment neither decreased the electrophoretic mobility of PKC-delta nor increased the amount of GTP bound to Ras, and PC-PLC was mitogenically active in BAC1.2F5 cells in which PKC-delta was downregulated by prolonged treatment with phorbol ester. PC-PLC mimicked CSF-1 action by elevating c-fos and junB mRNAs to 40% of the level induced by CSF-1; however, PC-PLC induced c-myc mRNA to only 5% of the level in CSF-1-stimulated cells. PC-PLC addition to CSF-1-dependent BAC1.2F5 clones that constitutively express c-myc increased [3H]thymidine incorporation to 86% of the level evoked by CSF-1 and supported slow growth in the absence of CSF-1. Therefore, PC-PLC is a component of a signal transduction pathway leading to transcription of c-fos and junB that collaborates with c-myc and is independent of PKC-delta and Ras activation.

Adolescent Trajectories of Aerobic Fitness and Adiposity as Markers of Cardiometabolic Risk in Adulthood.

Purpose: The aim of this study was to investigate whether adolescent growth trajectories of aerobic fitness and adiposity were associated with mid-adulthood cardiometabolic risk (CMR). Methods: Participants were drawn from the Saskatchewan Growth and Development Study (1963-1973). Adolescent growth trajectories for maximal aerobic capacity (absolute VO2 (AbsVO2)), skinfolds (SF), representing total body (Sum6SF) and central adiposity (TrunkSF), and body mass index (BMI) were determined from 7 to 17 years of age. In mid-adulthood (40 to 50 years of age), 61 individuals (23 females) returned for follow-ups. A CMR score was calculated to group participants as displaying either high or a low CMR. Multilevel hierarchical models were constructed, comparing the adolescent growth trajectories of AbsVO2, Sum6SF, TrunkSF, and BMI between CMR groupings. Results: There were no significant differences in the adolescent development of AbsVO2, Sum6SF, TrunkSF, and BMI between adult CMR groupings (p > 0.05). Individuals with high CMR accrued 62% greater adjusted total body fat percentage from adolescence to adulthood (p=0.03). Conclusions: Growth trajectories of adolescent aerobic fitness and adiposity do not appear to be associated with mid-adulthood CMR. Individuals should be encouraged to participate in behaviours that promote healthy aerobic fitness and adiposity levels throughout life to reduce lifelong CMR.

Regulation of mammalian cell membrane biosynthesis.

This review explores current information on the interrelationship between phospholipid biochemistry and cell biology. Phosphatidylcholine is the most abundant phospholipid and it biosynthesis has been studied extensively. The choline cytidylyltransferase regulates phosphatidylcholine production, and recent advances in our understanding of the mechanisms that govern cytidylyltransferase include the discovery of multiple isoforms and a more complete understanding of the lipid regulation of enzyme activity. Similarities between phosphatidylcholine formation and the phosphatidylethanolamine and phosphatidylinositol biosynthetic pathways are discussed, together with current insight into control mechanisms. Membrane phospholipid doubling during cell cycle progression is a function of periodic biosynthesis and degradation. Membrane homeostasis is maintained by a phospholipase A-mediated degradation of excess phospholipid, whereas insufficient phosphatidylcholine triggers apoptosis in cells.

Deregulated c-myc expression overrides IFN gamma-induced macrophage growth arrest.

Induction of c-myc gene expression is an essential response to growth promoting agents, including colony-stimulating factor 1 (CSF-1). Down regulation of c-myc expression occurs in response to a variety of negative growth regulators in many cell types. However, for many of these systems the causal link between c-myc down regulation and growth arrest remains to be established. Here we show for CSF-1-dependent BAC1.2F5 mouse macrophages that interferon-gamma (IFN gamma) results in a midlate G1 phase decrease of CSF-1-dependent c-myc mRNA and subsequent cell cycle arrest. Introduction of a deregulated c-myc gene into these cells, which prevents the IFN gamma-mediated decrease in c-myc expression, overrides the cell cycle arrest and restores CSF-1-dependent growth in the presence of the cytokine. This result contrasts with the macrophage growth arrest induced by cAMP elevation, which also suppresses c-myc expression, but is not overcome by a deregulated c-myc gene. These results show that inhibition of c-myc expression is an essential component in IFN gamma-mediated cell cycle arrest and demonstrates that distinct mechanisms contribute to IFN gamma- and cAMP-mediated growth arrest in macrophages.

Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells.

PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis.

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes.

An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.

The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine.

The mechanisms that account for the anti-proliferative properties of the biologically active lysophospholipid analog hexadecylphosphocholine (HexPC) were investigated in HL60 cells. HexPC inhibited the incorporation of choline into phosphatidylcholine and the pattern of accumulation of soluble choline-derived metabolites pinpointed CTP:phosphocholine cytidylyltransferase (CT) as the inhibited step in vivo. HexPC also inhibited recombinant CT in vitro. HexPC treatment led to accumulation of cells in G2/M phase, triggered DNA fragmentation and caused morphological changes associated with apoptosis. The supplementation of HexPC-treated cells with exogenous lysophosphatidylcholine (LPC) completely reversed the cytotoxic effects of HexPC and restored HL60 cell proliferation in the presence of the drug. LPC provided an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC. This result contrasted with the response of HL60 cells to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) where LPC overcame the cytotoxic effects but did not support continued cell proliferation. Morphological integrity, DNA stability and cell viability were maintained in cells treated with LPC plus either antineoplastic agent. Thus the inhibition of phosphatidylcholine biosynthesis at the CT step accounts for the cytotoxicity of both HexPC and ET-18-OCH3 which is overridden by providing an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC.

Actions of the brain-penetrating H2-antagonist zolantidine on histamine dynamics and metabolism in rat brain.

The effects of zolantidine, the first brain-penetrating H2-receptor antagonist, on the brain levels of histamine (HA) and the HA metabolite tele-methylhistamine (t-MH), the activity of histamine methyltransferase (HMT) and the brain HA turnover rates were investigated in rats. Zolantidine dimaleate (0.1 to 100 mg/kg, s.c.) had no effect on whole brain levels of HA or t-MH and no effect on brain HMT activity, when measured 30 min after administration. Furthermore, brain t-MH levels in pargyline-treated animals were unaffected by zolantidine (0.1 to 25 mg/kg), indicating the absence of an effect on brain HA turnover. In vitro, zolantidine was a potent competitive inhibitor of both brain and kidney HMT, with Ki values of 2.3 and 2.7 microM respectively. These results show that, despite the ability of zolantidine to inhibit HMT in vitro, large doses of this drug did not alter brain HA methylation or turnover in vivo, and they imply that blockade of post-synaptic H2-receptors does not change brain HA dynamics.

Histidine decarboxylase measurement in brain by 14CO2 trapping.

A method for measuring histidine decarboxylase (HDC) in crude rat brain homogenates was developed by modification of existing 14CO2-trapping methods. The addition of EDTA to tissue homogenates and assay buffer reduced non-enzymatic decarboxylation, and improved assay sensitivity and reliability. Addition of polyethylene glycol (molecular weight 300, PEG300) to the homogenizing buffer increased enzyme stability, permitting storage of crude homogenates. Studies of time course, tissue dilution and blanks showed that up to 8 mg of tissue could be assayed successfully with a 3.5-hr incubation. S-alpha-Fluoromethylhistidine (FMH) and alpha-hydrazinohistidine, specific inhibitors of HDC, induced concentration-dependent reductions of enzyme activity by up to 90%, whereas inhibitors of other decarboxylases had little or no effect. Kinetic studies of the enzyme in crude homogenates yielded Km and Vmax values similar to those found previously with other HDC methods, although a poor fit was found to a single enzyme model. When determined by the new method, the distribution of HDC in seven regions of the rat brain agreed well with previous results. The method is rapid, simple to perform, and requires no specialized equipment other than a scintillation counter.

Adenylate cyclase and ATPase activities in red cell membranes of patients and genetic carriers of Duchenne muscular dystrophy.

Basal adenylate cyclase activity was increased in red cell ghosts from both patients with Duchenne muscular dystrophy and their mothers when the activities were compared to proper age-matched controls. The activity of ATPase measured in the presence of Na+, K+, and Mg2+ was not found to be different in erythrocyte ghosts from Duchenne dystrophic patients, age-matched controls, or the mothers of Duchenne patients, and ouabain inhibited ATPase in ghosts to the same extent in all membrane preparations.

Using membrane stress to our advantage.

The nature of the bilayer motif coupled with the ability of lipids and proteins to diffuse freely through this structure is crucial to the viability of cells and their ability to compartmentalize domains contained therein. It seems surprising to find then that biological as well as model membranes exist in a dynamic state of mechanical stress. The stresses within such membranes are surprisingly large, typically reaching up to 50 atm (1 atm=101.325 kPa) at the core of the membrane and vary as a function of depth. The uneven distribution of lateral pressures within monolayer leaflets causes them to bend away from or towards the water interface. This can result in the formation of complex, self-assembled mesophases, many of which occur in vivo. Our knowledge of the principles underlying membrane mechanics has reached the point where we are now able to manipulate them and create nano-structures with reasonable predictability. In addition, they can be used both to explain and control the partitioning of amphipathic proteins on to membranes. The dependence of the dynamics of membrane-bound proteins and the chemical reactivity of amphipathic drug molecules on membrane stresses suggests that Nature itself takes advantage of this. Understanding and manipulating these internal forces will be a key element in creating self-assembled, biocompatible, nanoscale cell-like systems.

Coordination of membrane phospholipid synthesis with the cell cycle.

The formation of membrane phospholipid is coordinated with the cell cycle in a colony-stimulating factor 1-dependent macrophage cell line. Net phospholipid accumulation occurs as cells enter S phase and results from an interaction between cell cycle-dependent oscillations in the rates of phosphatidylcholine (PtdCho) biosynthesis and degradation. CTP:phosphocholine cytidylyltransferase (CT) is the rate-controlling step in PtdCho biosynthesis, and its activity is inhibited by cell cycle-dependent phosphorylation. CT phosphorylation is low in early G1, begins to rise in late G1, increases steadily through S and G2/M, and then declines precipitously as cells exit mitosis and reenter G1. CT activity increases in early G1 and then decreases steadily as the cells progress through late G1, S, and G2/M, reflecting the elevated phosphorylation state of the protein. Membrane phospholipid degradation is also periodic: the rate is rapid in G1, slows significantly during S phase, and accelerates again as cells reenter G1. The data support the hypothesis that CT dephosphorylation accelerates PtdCho synthesis in response to the increased membrane phospholipid degradation to maintain membrane phospholipid mass during G1 and that the periodic cessation of phospholipid degradation during S phase accounts for the transition to net membrane phospholipid accumulation. Inactivation of CT associated with hyperphosphorylation of the protein during G2/M likely plays a determining role in the cessation of net membrane accumulation prior to cell division.

Apoptosis triggered by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine is prevented by increased expression of CTP:phosphocholine cytidylyltransferase.

A HeLa cell line was constructed for the regulation of CTP:phosphocholine cytidylyltransferase (CCT) expression via a tetracycline-responsive promoter to test the role of CCT in apoptosis triggered by exposure of cells to the antineoplastic phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). Basal CCT expression in the engineered HeLa cell line was the same as in control HeLa cells lines, and CCT activity and protein were elevated 25-fold following 48 h of induction with doxycycline. Increased CCT expression prevented ET-18-OCH3-induced apoptosis. Acylation of exogenous lysophosphatidylcholine circumvented the requirement for CCT activity by providing an alternate route to phosphatidylcholine, and heightened CCT expression and lysophosphatidylcholine supplementation were equally effective in reversing the cytotoxic effect of ET-18-OCH3. Neither CCT overexpression nor lysophosphatidylcholine supplementation allowed the HeLa cells to proliferate in the presence of ET-18-OCH3, indicating that the cytostatic property of ET-18-OCH3 was independent of its effect on membrane phospholipid synthesis. These data provide compelling genetic evidence to support the conclusion that the interruption of phosphatidylcholine synthesis at the CCT step by ET-18-OCH3 is the primary physiological imbalance that accounts for the cytotoxic action of the drug.

Lipid activation of CTP:phosphocholine cytidylyltransferase is regulated by the phosphorylated carboxyl-terminal domain.

The role of the phosphorylated carboxyl-terminal domain of CTP:phosphocholine cytidylyltransferase (CT) in the regulation of enzyme activity was investigated by comparing the catalytic properties of wild-type CT to two mutant proteins with altered carboxyl-terminal phosphorylation domains. CT isolated from a baculovirus expression system was extensively phosphorylated at multiple sites in the carboxyl-terminal domain. The CT[S315A] mutant lacked a major CT phosphorylation site, and the carboxyl-terminal deletion mutant, CT[delta 312-367], was not phosphorylated. The higher activities of CT[delta 312-367] and CT[S315A] relative to CT were attributed to differences in the sensitivities of the enzymes to lipid activators. The rank order of the apparent Km values for activation by either phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol was CT > CT[S315A] > CT[delta 312-367]. In addition, CT exhibited negative cooperativity in its activation by phosphatidylcholine/oleic acid (nH = 0.64) and phosphatidylcholine/diacylglycerol (nH = 0.74) vesicles, whereas CT[delta 312-367] and CT[S315A] did not. These data support the concept that the phosphorylation of the CT carboxyl-terminal domain interferes with the activation of CT by lipid regulators.