Burrowing is a sensitive behavioural assay for monitoring general wellbeing during dextran sulfate sodium colitis in laboratory mice.

An impaired intestinal epithelial barrier is thought to be a major factor in the pathogenesis of human inflammatory bowel disease (IBD). IBD is frequently investigated by inducing a damaged barrier in murine models of colitis. This can be done by feeding mice with dextran sulfate sodium (DSS) polymers in their drinking water. Refinement measures should focus on alleviating unnecessary suffering during this probably painful condition. Appropriate parameters are needed to decide when to terminate the experiments. Our aim was to investigate whether a change in burrowing behaviour is a sensitive measure of animal welfare in murine models of colitis. Acute colitis was induced in C57BL/6 mice with 2.0% DSS over nine days. The burrowing test is based on the species-typical behaviour of mice to spontaneously displace items from tubes within their home cage. As a burrowing apparatus, a water bottle (250 mL, 150 mm length, 55 mm diameter) filled with 138-142 g of pellets of the animal's diet was used. The presence of intestinal inflammation as a result of acute DSS-induced colitis was confirmed by a decrease in body weight, colon length and an increase of murine endoscopic index of colitis severity, histological score and spleen weight in the group receiving DSS as compared with the control group. An onset of intestinal inflammation correlated with a significant decrease in burrowing behaviour (P < 0.05). Altered adrenal gland histology indicated stress as a result of acute colitis. Our findings provide evidence that changes of spontaneous burrowing behaviour correlate with the onset of inflammation in acute DSS-induced colitis.

Evaluation of moldable, in situ hardening calcium phosphate bone graft substitutes.

OBJECTIVE: Moldable in situ self-stabilizing and hardening bone graft materials facilitate handling and may be suitable for membrane-free bone regeneration methods. This study aimed to compare two moldable synthetic calcium phosphate materials in a rabbit calvarial defect model. METHOD: In 12 New Zealand white rabbits, four evenly distributed 6 mm diameter defects were drilled in the calvarial bone. Three filler materials were randomly applied to 48 defects: an in situ hardening polylactide-coated ß-tricalcium phosphate (TCP), an in situ hardening polylactide-coated biphasic calcium phosphate (BCP), and a granular deproteinized bovine bone matrix (DBBM, positive control). One defect remained untreated and served as a negative control. Six animals were sacrificed after 4 weeks, and the remaining animals were sacrificed after 16 weeks. Biocompatibility, bone graft substitute integration and resorption, bone formation, defect bridging, and height of reconstructed hard tissue were assessed histologically and histomorphometrically. RESULTS: All tested materials showed good biocompatibility. Semi-quantitative analysis and pair-wise comparison suggested that BCP was more efficient in centripetal bone formation when compared with TCP. After 4 weeks, significantly more bone had formed in the defects treated with either TCP or BCP materials compared with the untreated sites. BCP and DBBM did not show macroscopic signs of degradation, whereas the TCP material was partially resorbed after 16 weeks. Otherwise, no major differences were detected between the three materials. CONCLUSION: The moldable, synthetic calcium phosphates are safe and suitable bone graft substitutes with outcomes that are comparable to the control material.

Bone regeneration in the presence of a synthetic hydroxyapatite/silica oxide-based and a xenogenic hydroxyapatite-based bone substitute material.

OBJECTIVES: A comparison of synthetic hydroxyapatite/silica oxide, xenogenic hydroxyapatite-based bone substitute materials with empty control sites in terms of bone regeneration enhancement in a rabbit calvarial four non-critical-sized defect model. METHODS: In each of six rabbits, four bicortical calvarial bone defects were generated. The following four treatment modalities were randomly allocated: (1) empty control site, (2) synthetic hydroxyapatite/silica oxide-based (HA/SiO) test granules, (3) xenogenic hydroxyapatite -based granules, (4) synthetic hydroxyapatite/silica oxide -based (HA/SiO) test two granules. The results of the latter granules have not been reported due to their size being three times bigger than the other two granule types. After 4 weeks, the animals were sacrificed and un-decalcified sections were obtained for histological analyses. For statistical analysis, the Kruskal-Wallis test was applied (P<0.05). RESULTS: Histomorphometric analysis showed an average area fraction of newly formed bone of 12.32±10.36% for the empty control, 17.47±6.42% for the xenogenic hydroxyapatite -based granules group, and 21.2±5.32% for the group treated with synthetic hydroxyapatite/silica oxide -based granules. Based on the middle section, newly formed bone bridged the defect to 38.33±37.55% in the empty control group, 54.33±22.12% in the xenogenic hydroxyapatite -based granules group, and to 79±13.31% in the synthetic hydroxyapatite/silica oxide -based granules group. The bone-to-bone substitute contact was 46.38±18.98% for the xenogenic and 59.86±14.92% for the synthetic hydroxyapatite/silica oxide-based granules group. No significant difference in terms of bone formation and defect bridging could be detected between the two bone substitute materials or the empty defect. CONCLUSION: There is evidence that the synthetic hydroxyapatite/silica oxide granules provide comparable results with a standard xenogenic bovine mineral in terms of bone formation and defect bridging in non-critical size defects.

Genetic analysis of the additional sex combs locus of Drosophila melanogaster.

Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.

Lithium clearance: modification by the loop of Henle in man.

1. The contribution of Li+ reabsorption in the loop of Henle to lithium clearance (CLi) and the possible mechanism(s) involved were assessed in healthy volunteers. Four mechanisms were considered: (a) passive reabsorption in the thin ascending limb, (b) solvent drag in the thin descending limb, (c) the Na+, K+, 2Cl- transporter in the thick ascending limb and (d) paracellular movement in the thick ascending limb. 2. Since alterations in the corticomedullary osmolal concentration gradient produced by fluid restriction (500 ml day-1) and subsequent water loading (15 ml kg-1) did not affect either CLi (28.5 +/- 2.1 vs. 28.2 +/- 1.9 ml min-1) or fractional lithium clearance (FELi; 23.5 +/- 2.0 vs. 23.0 +/- 1.9%), it is unlikely that substantial Li+ reabsorption occurs in the thin limbs by either passive movement or solvent drag. 3. Increasing plasma Li+ with unchanged plasma Na+ in salt-replete volunteers was associated with only small reductions in CLi (32.8 +/- 1.3 ml min-1, P less than 0.05) and FELi (27.3 +/- 1.8 vs. 25.3 +/- 2.0%, P less than 0.05). This suggests that substantial Li+ reabsorption on the Na+, K+, 2Cl- transporter does not occur. 4. Bumetanide increased FELi in salt-depleted (LS) and salt-replete (HS) volunteers and abolished the pre-diuretic difference in FELi between salt intakes (LS, 16.6 +/- 1.5 vs. 38.7 +/- 2.3%, P less than 0.001; HS, 30.1 +/- 1.5 vs. 40.5 +/- 2.0%, P less than 0.001). Changes in CPO4 and CHCO3 were not detected. Acetazolamide produced comparable increases in FELi (LS, 16.6 +/- 1.5 vs. 38.7 +/- 2.2%, P less than 0.001; HS, 30.1 +/- 1.5 vs. 43.1 +/- 2.4%, P less than 0.01); and CPO4 and CHCO3 were increased. When tubular flow to the loop of Henle was increased by acetazolamide, the bumetanide-induced increases in FELi were reduced (LS, 38.7 +/- 2.2 vs. 48.7 +/- 2.3%, P less than 0.001; HS, 43.1 +/- 2.4 vs. 48.1 +/- 2.6%, P less than 0.001). 5. These data are consistent with the view that (a) Li+ is reabsorbed by a bumetanide-sensitive mechanism in the loop of Henle, (b) approximately 20 and 10% of the filtered load, respectively, is reabsorbed in the loop in salt-depleted and salt-replete volunteers, (c) flow-dependent, voltage-driven paracellular movement in the thick ascending limb is the likely mechanism and (d) this mechanism could account for the difference in Li+ reabsorption between low and high salt intakes.

An investigation into the role of photosynthesis in regulating ATP levels and rates of h efflux in isolated meosphyll cells.

Aerated and stirred 10-ml suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used for simultaneous measurements of net H(+) efflux and steady-state ATP levels.Initial rates of medium acidification indicated values for H(+) efflux in the light and dark of 0.66 and 0.77 nanomoles H(+)/10(6) cells per minute, respectively. When the medium pH was maintained at 6.5, with a pH-stat apparatus, rates of H(+) efflux remained constant. Darkness or DCMU, however, stimulated H(+) efflux by 100% or more. Darkness increased ATP levels by 33% and a switch from dark to light reduced ATP levels by 31%. In the absence of aeration, illumination prevented the accumulation of respiratory CO(2) and the buffering capacity of the medium was about 50% less than that found in the nonilluminated nonaerated medium. As a result, rates of pH decline were similar even though the dark rate of H(+) efflux was approximately 50% greater.Proposals that photosynthesis stimulates H(+) efflux are based on changes in the rate of pH decline. The present data indicate that photosynthesis inhibits H(+) efflux and that changes in rates of pH decline should not be equated with changes in the rate of H(+) efflux.

Inhibition of purified rat liver glutathione S-transferase isozymes by diuretic drugs.

Seven soluble rat liver glutathione S-transferase isozymes were isolated and the inhibition of these isozymes by selected diuretics was investigated using 1-chloro-2,4-dinitrobenzene as substrate. All isozymes were inhibited to some extent under the experimental conditions used, but there was significant isozyme dependent selectivity of inhibition. The greatest inhibitory effect (over 80%) was found when the phenoxyacetic acid diuretics and indacrynic acid were incubated with glutathione S-transferase 3-3, 3-4 and 4-4. The sulphamoylbenzoic acid diuretics, furosemide and bumetanide, were found to have a lesser effect on the isozymes studies. As glutathione S-transferase are thought to play an important protective role in the various tissues of animals and man, by catalysing the glutathione conjugation of electrophilic drugs and drug metabolites, their inhibition may be toxicologically important.

Inhibition of purified glutathione S-transferases by indomethacin.

Soluble rat liver glutathione S-transferases have been purified and a previously undescribed peak was observed. This peak contained glutathione S-transferase activity which was extensively inhibited by indomethacin. Glutathione conjugation of 1-chloro-2,4-dinitrobenzene by this isozyme, designated glutathione S-transferase VII, was inhibited 44 and 68% at indomethacin concentrations of 0.20 and 1.00 microM, respectively. The other six basic glutathione S-transferase isozymes were relatively unaffected by low concentrations of indomethacin. The pharmacological significance of this inhibition by indomethacin is largely dependent on the role of the glutathione S-transferase VII in leukotriene synthesis.

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3.

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.