Intestinal permeability and contractility in murine colitis.

We developed an in vitro organ bath method to measure permeability and contractility simultaneously in murine intestinal segments. To investigate whether permeability and contractility are correlated and influenced by mucosal damage owing to inflammation, BALB/c mice were exposed to a 10% dextran sulphate sodium (DSS) solution for 8 days to induce colitis. The effect of pharmacologically induced smooth muscle relaxation and contraction on permeability was tested in vitro. Regional permeability differences were observed in both control and 10% DSS-treated mice. Distal colon segments were less permeable to 3H-mannitol and 14C-PEG 400 molecules compared with proximal colon and ileum. Intestinal permeability in control vs. 10% DSS mice was not altered, although histologic inflammation score and IFN-gamma pro-inflammatory cytokine levels were significantly increased in proximal and distal colon. IL-1beta levels were enhanced in these proximal and distal segments, but not significantly different from controls. Any effect of pharmacologically induced contractility on intestinal permeability could not be observed. In conclusion, intestinal permeability and contractility are not correlated in this model of experimentally induced colitis in mice. Although simultaneous measurement in a physiological set-up is possible, this method has to be further validated.     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20-24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-alpha 1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34-48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of alpha-amanitin in the culture media at 0 h. The inhibiting effect of alpha-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.     

Molecular mechanisms of type III hyperlipoproteinemia: The contribution of the carboxy-terminal domain of ApoE can account for the dyslipidemia that is associated with the E2/E2 phenotype

Apolipoprotein E2, which has an R158 for C substitution, has reduced affinity for the LDL receptor and is associated with type III hyperlipoproteinemia in humans. Consistent with these observations, we have found that following adenovirus-mediated gene transfer, full-length apoE2 aggravates the hypercholesterolemia and induces hypertriglyceridemia in E-deficient mice and induces combined hyperlipidemia in C57BL/6 mice. Unexpectedly, the truncated apoE2-202 form that has an R158 for C substitution when expressed at levels similar to those of the full-length apoE2 normalized the cholesterol levels of E-deficient mice without induction of hypertriglyceridemia. The apoE2 truncation increased the affinity of POPC-apoE particles for the LDL receptor, and the full-length apoE2 had a dominant effect in VLDL triglyceride secretion. Hyperlipidemia in normal C57BL/6 mice was prevented by coinfection with equal doses of each, the apoE2 and the apoE2-202-expressing adenoviruses, indicating that truncated apoE forms have a dominant effect in remnant clearance. Hypertriglyceridemia was completely corrected by coinfection of mice with an adenovirus-expressing wild-type lipoprotein lipase, whereas an inactive lipoprotein lipase had a smaller effect. The findings suggest that the apoE2-induced dyslipidemia is not merely the result of substitution of R158 for C but results from increased secretion of a triglyceride-enriched VLDL that cannot undergo lipolysis, inhibition of LpL activity, and impaired clearance of chylomicron remnants. Infection of E(-)(/)(-)xLDLr(-)(/)(-) double-deficient mice with apoE2-202 did not affect the plasma cholesterol levels, and also did not induce hypertriglyceridemia. In contrast, apoE2 exacerbated the hypercholesterolemia and induced hypertriglyceridemia, suggesting that the LDL receptor is the predominant receptor in remnant clearance.     

High-fat diets rich in medium- versus long-chain fatty acids induce distinct patterns of tissue specific insulin resistance

Excess dietary long-chain fatty acid (LCFA) intake results in ectopic lipid accumulation and insulin resistance. Since medium-chain fatty acids (MCFA) are preferentially oxidized over LCFA, we hypothesized that diets rich in MCFA result in a lower ectopic lipid accumulation and insulin resistance compared to diets rich in LCFA. Feeding mice high-fat (HF) (45% kcal fat) diets for 8 weeks rich in triacylglycerols composed of MCFA (HFMCT) or LCFA (HFLCT) revealed a lower body weight gain in the HFMCT-fed mice. Indirect calorimetry revealed higher fat oxidation on HFMCT compared to HFLCT (0.011.0±0.0007 vs. 0.0096±0.0015 kcal/g body weight per hour, P<.05). In line with this, neutral lipid immunohistochemistry revealed significantly lower lipid storage in skeletal muscle (0.05±0.08 vs. 0.30±0.23 area%, P <.05) and in liver (0.9±0.4 vs. 6.4±0.8 area%, P<.05) after HFMCT vs. HFLCT, while ectopic fat storage in low fat (LF) was very low. Hyperinsulinemic euglycemic clamps revealed that the HFMCT and HFLCT resulted in severe whole body insulin resistance (glucose infusion rate: 53.1±6.8, 50.8±15.3 vs. 124.6±25.4 μmol min⁻¹ kg⁻¹, P<.001 in HFMCT, HFLCT and LF-fed mice, respectively). However, under hyperinsulinemic conditions, HFMCT revealed a lower endogenous glucose output (22.6±8.0 vs. 34.7±8.5 μmol min⁻¹ kg⁻¹, P<.05) and a lower peripheral glucose disappearance (75.7±7.8 vs. 93.4±12.4 μmol min⁻¹ kg⁻¹, P<.03) compared to HFLCT-fed mice. In conclusion, both HF diets induced whole body insulin resistance compared to LF. However, the HFMCT gained less weight, had less ectopic lipid accumulation, while peripheral insulin resistance was more pronounced compared to HFLCT. This suggests that HF-diets rich in medium- versus long-chain triacylglycerols induce insulin resistance via distinct mechanisms.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Energy Expenditure and Metabolic Changes of Free-Flying Migrating Northern Bald Ibis

Many migrating birds undertake extraordinary long flights. How birds are able to perform such endurance flights of over 100-hour durations is still poorly understood. We examined energy expenditure and physiological changes in Northern Bald Ibis Geronticus eremite during natural flights using birds trained to follow an ultra-light aircraft. Because these birds were tame, with foster parents, we were able to bleed them immediately prior to and after each flight. Flight duration was experimentally designed ranging between one and almost four hours continuous flights. Energy expenditure during flight was estimated using doubly-labelled-water while physiological properties were assessed through blood chemistry including plasma metabolites, enzymes, electrolytes, blood gases, and reactive oxygen compounds. Instantaneous energy expenditure decreased with flight duration, and the birds appeared to balance aerobic and anaerobic metabolism, using fat, carbohydrate and protein as fuel. This made flight both economic and tolerable. The observed effects resemble classical exercise adaptations that can limit duration of exercise while reducing energetic output. There were also in-flight benefits that enable power output variation from cruising to manoeuvring. These adaptations share characteristics with physiological processes that have facilitated other athletic feats in nature and might enable the extraordinary long flights of migratory birds as well.