Micrococcal nuclease does not substantially bias nucleosome mapping

We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8-10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4-5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.     

Dietary iron loading does not influence biliary iron excretion in rats

The effect of dietary iron loading on biliary iron excretion was investigated with male Wistar rats aged 6 wk. The rats were fed purified diets with either 174 or 1740 mg FeSO₄. 7H₂O/kg diet and demineralized water for 6 wk. Blood haemoglobin, hematocrit, and iron concentrations in kidney and heart were not affected and iron concentrations in liver, spleen, and tibia were significantly raised after feeding the high-iron diet. The high-iron diet did not raise biliary iron excretion, suggesting that biliary iron excretion does not play an important role in regulating iron metabolism in rat after dietary iron loading.     

Heat shock does not attenuate low-frequency fatigue.

Whole-body hyperthermia or heat shock confers protection to myocardial contractility against reperfusion-induced injury. The purpose of this study was to determine whether heat shock could provide similar protection to skeletal muscle contractility against low-frequency fatigue. Male Sprague-Dawley rats (6 rats/group) were heat shocked at 41.5 degrees C for 15 min either 24 h or 4 days prior to fatiguing stimulation to compare the contractile responses of the plantaris muscle with those of a nonheated group. Both 24 h and 4 days after heat shock, the 72-kDa heat shock protein (HSP72) was elevated above control levels. There were no differences between the heat-shocked and non-heat-shocked animals in measures of contractility prior to fatiguing contractions or in resistance to fatigue. Heat-shock preconditioning did not lead to improved postfatigue force recovery above control responses and, in fact, delayed the recovery of force. This study does not support the use of heat-shock therapy to improve skeletal muscle contractile performance under fatiguing conditions.     

PrP(106-126) does not interact with membranes under physiological conditions

Transmissible spongiform encephalopathies are neurodegenerative diseases characterized by the accumulation of an abnormal isoform of the prion protein PrP^Sc. Its fragment 106-126 has been reported to maintain most of the pathological features of PrP^Sc, and a role in neurodegeneration has been proposed based on the modulation of membrane properties and channel formation. The ability of PrP^Sc to modulate membranes and/or form channels in membranes has not been clearly demonstrated; however, if these processes are important, peptide-membrane interactions would be a key feature in the toxicity of PrP^Sc. In this work, the interaction of PrP(106-126) with model membranes comprising typical lipid identities, as well as more specialized lipids such as phosphatidylserine and GM1 ganglioside, was examined using surface plasmon resonance and fluorescence methodologies. This comprehensive study examines different parameters relevant to characterization of peptide-membrane interactions, including membrane charge, viscosity, lipid composition, pH, and ionic strength. We report that PrP(106-126) has a low affinity for lipid membranes under physiological conditions without evidence of membrane disturbances. Membrane insertion and leakage occur only under conditions in which strong electrostatic interactions operate. These results support the hypothesis that the physiological prion protein PrP^C mediates PrP(106-126) toxic effects in neuronal cells.     

PTEN does not modulate GLUT4 translocation in rat adipose cells under physiological conditions.

PTEN is a 3'-inositol lipid phosphatase that dephosphorylates products of PI 3-kinase. Since PI 3-kinase is required for many metabolic actions of insulin, we investigated the role of PTEN in insulin-stimulated translocation of GLUT4. In control rat adipose cells, we observed a approximately 2-fold increase in cell surface GLUT4 upon maximal insulin stimulation. Overexpression of wild-type PTEN abolished this response to insulin. Translocation of GLUT4 in cells overexpressing PTEN mutants without lipid phosphatase activity was similar to that observed in control cells. Overexpression of PTEN-CBR3 (mutant with disrupted membrane association domain) partially impaired translocation of GLUT4. In Cos-7 cells, overexpression of wild-type PTEN had no effect on ERK2 phosphorylation in response to acute insulin stimulation. However, Elk-1 phosphorylation in response to chronic insulin treatment was significantly decreased. Thus, when PTEN is overexpressed, both its lipid phosphatase activity and subcellular localization play a role in antagonizing metabolic actions of insulin that are dependent on PI 3-kinase but independent of MAP kinase. However, because translocation of GLUT4 in cells overexpressing a dominant inhibitory PTEN mutant (C124S) was similar to that of control cells, we conclude that endogenous PTEN may not modulate metabolic functions of insulin under normal physiological conditions.     

SAA does not induce cytokine production in physiological conditions

SAA has been shown to have potential proinflammatory properties in inflammatory diseases such as atherosclerosis. These include induction of tumor necrosis factor α, interleukin-6, and monocyte chemoattractant protein 1 in vitro. However, concern has been raised that these effects might be due to use of recombinant SAA with low level of endotoxin contaminants or its non-native forms. Therefore, physiological relevance has not been fully elucidated. In this study, we investigated the role of SAA in the production of inflammatory cytokines. Stimulation of mouse monocyte J774 cells with lipid-poor recombinant human SAA and purified SAA derived from cardiac surgery patients, but not ApoA-I and ApoA-II, elicited pro-inflammatory cytokines like granulocyte colony stimulating factor (G-CSF). However, HDL-associated SAA failed to stimulate production of these cytokines. Using neutralizing antibodies against toll like receptor (TLR) 2 and 4, we could evaluate that TLR 2 is responsible for G-CSF production by lipid-poor SAA. To confirm these data in vivo, we expressed mouse SAA in SAA deficient C57BL/6 mice using an adenoviral vector. G-CSF was identically expressed in SAA-Adenoviral infected mice as well as in control null-Adenoviral mice at the early time points (4–8 h) and could not be detected in plasma 24 h after infection when plasma SAA levels were maximally elevated, indicating that adenoviral vector rather than SAA affected G-CSF levels. Taken together, our findings suggest that lipid-poor SAA, but not HDL-associated SAA, stimulates G-CSF production and this stimulation is mediated through TLR 2 in J774 cells. However, its physiological role in vivo remains ambiguous.     

26-Hydroxylation of 1alpha,25-dihydroxyvitamin D3 does not occur under physiological conditions

The 26-hydroxylation of 1alpha,25-dihydroxyvitamin D3 in rats in vitro and in vivo was studied under physiological conditions. Incubation of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 with rat kidney or rat liver homogenate showed formation of a metabolite that was identified as 1alpha,25(S),26-trihydroxy-[26,27-3H]vitamin D3 by comigration on three different HPLC systems and a periodate cleavage reaction. This metabolite was not generated by hydroxylation of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 itself but by an enzymatic conversion of a precursor that was formed nonenzymatically in substantial amounts upon storage of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 in ethanol at -20 degrees C under argon for more than three weeks. An in vivo metabolism study in rats dosed with a physiological dose of 1alpha,25-dihydroxy-[26,27-3H]vitamin D3 confirmed the absence of 26-hydroxylation of the hormone. As expected at 6 h postinjection of purified 1alpha,25-dihydroxy-[26,27-3H]vitamin D3, 1alpha,24(R),25-trihydroxy-[26,27-3H]vitamin D3, as well as traces of (23S,25R)-1alpha,25-dihydroxy-[3H]vitamin D3-lactone were detected and identified on straight phase and reverse phase HPLC in serum, kidney, and liver.     

Continuum damage mechanics (CDM) modelling demonstrates that ligament fatigue damage accumulates by different mechanisms than creep damage

Ligaments can be subjected to creep and fatigue damage when loaded to higher than normal stresses due to injury of a complementary joint restraint. Continuum damage mechanics (CDM) assumes that diffuse damage accumulates in a material, thereby reducing the effective cross-sectional area and leading to eventual rupture. The objective of this study was to apply CDM modelling to ligament creep and fatigue to reveal mechanisms of damage. Fatigue was modelled by cyclically varying the stress in the creep model. A few novel approaches were used. First, area reduction was not assumed equal to modulus reduction; thus, allowing damaged fibres to potentially contribute to load-bearing through the extracellular matrix. Modulus ratio was related to area reduction using residual strength. Second, damage rate was not assumed constant but rather was determined directly from the modulus ratio change with respect to time. Third, modulus ratio was normalized to maximum modulus to avoid artificial calculation of negative damage. With this approach, the creep time-to-rupture was predicted with -4% error at 60% UTS and -13% error at 30% UTS. At 15% UTS, no test was undertaken experimentally for a duration as long as the 24 days predicted theoretically. Oscillating the time-dependent damage in the creep model could not completely explain the fatigue behaviour because the fatigue time-to-rupture was predicted with over 1300% error at all stresses. These results suggest that a cycle-dependent damage mechanism, in addition to a time-dependent one, was responsible for fatigue rupture. Cycle-dependent damage may be an important consideration for rehabilitation activities following injury of a complementary ligament restraint.     

The influence of exogenous cross-linking and compressive creep loading on intradiscal pressure

This study involves a biomechanical evaluation of a prospective injectable treatment for degenerative discs. The high osmolarity of the non-degenerated nucleus pulposus attracts water contributing to the hydrostatic behavior of the tissue. This intradiscal pressure is known to drop as fluid is exuded from the matrix due to compressive loading. The objective of this study was to compare the changes in intradiscal pressure in control and genipin cross-linked intervertebral discs. Thirty bovine lumbar motion segments were randomly divided into a phosphate-buffered saline control group and a 0.33% genipin group and soaked at room temperature for 2 days. A needle pressure sensor was held in the center of the disc while short-term and static creep compressive loads were applied. The control group demonstrated a 25% higher average intradiscal pressure compared to genipin-treated discs under 750 N compressive load (p=0.029). Depressurization during static compressive creep was 56% higher in the control than in the genipin group (p=0.014). These results suggest cross-linking induced changes in the poroelastic properties of the involved tissues affected the mechanics of compressive load support in the disc with lower levels of nucleus pressure, a corresponding decrease in the elastic expansion of the annulus, and an increased axial compressive loading of the inner and outer annulus tissues. It is possible that concurrent changes in hydraulic permeability and proteoglycan retention known to be associated with genipin cross-linking were also contributors to poroelastic changes. Reduction of peak pressures and moderation of pressure fluctuations could be beneficial relative to discogenic pain.     

Creep does not contribute to fatigue in bovine trabecular bone

In both cortical and trabecular bone loaded in fatigue, the stress-strain loops translate along the strain axis. Previous studies have suggested that this translation is the result of creep associated with the mean stress applied in the fatigue test. In this study, we measured the residual strrain (corresponding to the translation of the stress-strain loops) in fatigue tests on bovine trabecular bone and compared it to an upper bound estimate of the creep strain in each test. Our results indicate that the contribution of creep to the translation of the stress-strain loops is negligible in bovine trabecular bone. These results, combined with models for fatigue in lower density bone, suggest that that creep does not contribute to the fatigue of normal human bone. Creep may make a significant contribution to fatigue in low-density osteoporotic bone in which trabeculae have resorbed, reducing the connectivity of the trabecular structure.     

Sexual selection does not influence minisatellite mutation rate

Background  

Moller and Cuervo report a significant trend between minisatellite mutation rate and the frequency of extra-pair copulations in birds. This is interpreted as evidence that the high rate of evolution demanded by sexual selection has itself selected for a higher mutation rate in species where selection is strongest. However, there are good a priori reasons for believing that their method of calculating minisatellite mutation rates will be highly error prone and a poor surrogate measure of the evolutionary rate of genes. I therefore attempted to replicate their results using both their data and an independent data set based on papers they failed to locate.     

Supraspinal fatigue does not explain the sex difference in muscle fatigue of maximal contractions.

Young women are less fatigable than young men for maximal and submaximal contractions, but the contribution of supraspinal fatigue to the sex difference is not known. This study used cortical stimulation to compare the magnitude of supraspinal fatigue during sustained isometric maximal voluntary contractions (MVCs) performed with the elbow flexor muscles of young men and women. Eight women (25.6 +/- 3.6 yr, mean +/- SD) and 9 men (25.4 +/- 3.8 yr) performed six sustained MVCs (22-s duration each, separated by 10 s). Before the fatiguing contractions, the men were stronger than the women (75.9 +/- 9.2 vs. 42.7 +/- 8.0 N.m; P < 0.05) in control MVCs. Voluntary activation measured with cortical stimulation before fatigue was similar for the men and women during the final control MVC (95.7 +/- 3.0 vs. 93.3 +/- 3.6%; P > 0.05) and at the start of the fatiguing task (P > 0.05). By the end of the six sustained fatiguing MVCs, the men exhibited greater absolute and relative reductions in torque (65 +/- 3% of initial MVC) than the women (52 +/- 9%; P < 0.05). The increments in torque (superimposed twitch) generated by motor cortex stimulation during each 22-s maximal effort increased with fatigue (P < 0.05). Superimposed twitches were similar for men and women throughout the fatiguing task (5.5 +/- 4.1 vs. 7.3 +/- 4.7%; P > 0.05), as well as in the last sustained contraction (7.8 +/- 5.9 vs. 10.5 +/- 5.5%) and in brief recovery MVCs. Voluntary activation determined using an estimated control twitch was similar for the men and women at the start of the sustained maximal contractions (91.4 +/- 7.4 vs. 90.4 +/- 6.8%, n = 13) and end of the sixth contraction (77.2 +/- 13.3% vs. 73.1 +/- 19.6%, n = 10). The increase in the area of the motor-evoked potential and duration of the silent period did not differ for men and women during the fatiguing task. However, estimated resting twitch amplitude and the peak rates of muscle relaxation showed greater relative reductions at the end of the fatiguing task for the men than the women. These results indicate that the sex difference in fatigue of the elbow flexor muscles is not explained by a difference in supraspinal fatigue in men and women but is largely due to a sex difference of mechanisms located within the elbow flexor muscles.     

Damage evolution in acetabular replacements under long-term physiological loading conditions

Damage development in cemented acetabular replacements has been studied in bovine pelvic bones under long-term physiological loading conditions, including normal walking, stair climbing and a combined block loading with representative routine activities. The physiological loading conditions were achieved using a specially designed hip simulator for fixation endurance testing. Damage was detected and monitored using micro-CT scanning at regular intervals of the experiments, and verified by microscopic studies post testing. The results show that debonding at the bone–cement interface defined the failure of cement fixation in all cases, and debondings initiated near the dome of the acetabulum in the superior–posterior quadrant, consistent with the high-stress region identified from the finite element analysis of implanted acetabular models Zant, N.P., Heaton-Adegbile, P., Hussell, J.G., Tong, J., 2008b. In-vitro fatigue failure of cemented acetabular replacements—a hip simulator study. Journal of Biomechanical Engineering, Transactions of the ASME, 130, 021019-1–9]; [Tong, J., Zant, N.P., Wang, J-Y., Heaton-Adegbile, P., Hussell, J.G., 2008. Fatigue in cemented acetabulum. International Journal of Fatigue, 30(8), 1366–1375].     

Chromium picolinate does not produce chromosome damage in CHO cells

Chromium picolinate (CrPic, Chromax) is a dietary supplement that has been commercially available for the past two decades. CrPic has potential benefits for reducing insulin dependence in diabetics by increasing sensitivity of insulin receptors and in stimulating insulin binding. In this study, CrPic was tested for its ability to produce chromosomal aberrations in vitro using Chinese hamster ovary K1 (CHO) cells. CHO cells were exposed to a range of cytotoxic to non-cytotoxic concentrations of CrPic for 4 or 20h in the absence of metabolic (S9) activation or for 4h in the presence of S9 activation. CrPic was solubilized with dimethyl sulfoxide (DMSO) to attain the highest possible solubility for maximizing the test doses. Cells were treated with 96.25, 192.5, 385 or 770 microg/mL of CrPic for 4 h in the presence of S9 activation, and for 4 or 20 h in the absence of S9 activation. A distinct precipitate of CrPic was evident in the cell culture medium at 770 microg/mL, which was the highest dose tested. Results showed no statistically significant increases in structural or numerical chromosome aberrations were produced at any test dose level with CrPic in 4-h treatments up to a precipitating dose of 770 microg/mL in either the presence or absence of S9 activation. Additionally no aberrations were observed up to 385 microg/mL (the maximum analyzable dose) following treatment for 20 h in the absence of S9 activation. The percentage of cells with structural or numerical aberrations in CrPic treated cultures was not statistically different (p>0.05) from that quantified in controls at any dose level. The absence of significant differences from control levels demonstrates that CrPic did not induce structural or numerical chromosome aberrations up to doses that were insoluble in the culture medium.