Differential degradation of variant medium-chain acyl-CoA dehydrogenase by the protein quality control proteases Lon and ClpXP

The coordinated activities of chaperones and proteases that supervise protein folding and degradation are important factors for deciding the fate of proteins whose folding is impaired by missense variations. We have studied the role of Lon and ClpXP proteases in handling of wild-type and a folding-impaired disease-associated variant (R28C) of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). Using an Escherichia coli model system, we co-overexpressed the MCAD variants and the respective proteases at two conditions: at 31 degrees C where R28C MCAD protein folds partially and at 37 degrees C where it misfolds and aggregates. Co-overexpression of Lon protease considerably accelerated the degradation rate of a pool of R28C variant MCAD synthesised during a 30min pulse and counteracted accumulation of aggregates at 37 degrees C, whereas increasing the amounts of ClpXP protease had no clear effect. Co-overexpression of either Lon or ClpXP protease markedly decreased the steady state levels of both wild-type and R28C mutant MCAD at 37 degrees C but not at 31 degrees C. Our results suggest that Lon is more efficient than ClpXP in elimination of non-native MCAD protein conformations, and accordingly, that Lon can recognise a broader spectrum of MCAD protein conformations.     

Effect of smooth muscle tone on morphometry and residual strain in rat duodenum, jejunum and ileum

It is difficult to measure gastrointestinal smooth muscle (SM) tone except in sphincter regions. Since tone affects the biomechanical properties, the aim of the present study was to evaluate intestinal SM tone by studying the morphometry and biomechanical properties with and without muscle tone. Circumferential rings of 0.8-1mm in width were cut from the rat duodenum, jejunum and ileum. Sectors were obtained by cutting the rings opposite to the mesentery. The rings and the sectors were immersed in physiological Krebs solution in order to maintain the tone and into Krebs solution without Ca(++) and with EGTA to abolish the tone. The circumferences, area, the circularity and residual strain of the mucosal and serosal surfaces, opening angle, and opening angle tone/non-tone ratio were measured or computed. The tone affects the opening angle and residual strain in the intestinal sectors. The opening angle in the tissue sectors with tone was smaller (P<0.05) than those without tone in all three segments. The opening angle tone/non-tone ratio was 0.40+/-0.05, 0.43+/-0.06 and 0.36+/-0.11 for duodenum, jejunum and ileum, respectively, and did not differ among the three intestinal segments. The residual strain between sectors with and without SM tone differed in duodenal and jejunal mucosa and in the serosa of all three segments (P<0.05). The intestinal rings with tone showed axial variation for luminal area (P<0.001), for wall area (P<0.05), and for the mucosal and serosal residual strains (P<0.05). In conclusion, the intestinal mechanical properties are affected by intestinal SM tone. The tone can be evaluated by measuring the opening angle and residual strains of sectors in intestinal segments with and without SM tone.     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.     

Adsorption of ethylenediaminetetraacetic dianhydride modified oxalate decarboxylase on calcium oxalate

We used ethylenediaminetetraacetic acid dianhydride (EDTAD) to modify oxalate decarboxylase (OXDC) to improve its adsorption on calcium oxalate stones. The modified sites were identified by Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and the adsorption mechanism of the EDTAD-modified OXDC on calcium oxalate (CaOx) was investigated. We investigated adsorption time, initial enzyme concentration, temperature and solution pH on the adsorption process. Data were analyzed using kinetics, thermodynamics and isotherm adsorption models. UPLC-MS showed that EDTAD was attached to OXDC covalently and suggested that the chemical modification occurred at both the free amino of the side chain and the α-NH₂ of the peptide. The adsorption capacity of the EDTAD-OXDC on calcium oxalate was 53.37% greater than that of OXDC at the initial enzyme concentration of 5 mg/ml, pH = 7.0, at 37° C. The modified enzyme (EDTAD-OXDC) demonstrated improved oxalate degradation activity at pH 4.5?6.0. Kinetic data fitting analysis suggested a pseudo second order kinetic model. Estimates of the thermodynamic parameters including ΔG⁰, ΔH⁰ and ΔS⁰ of the adsorption process showed it to be feasible, spontaneous and endothermic. Isotherm data fitting analysis indicated that the adsorption process is reduced to monolayer adsorption at a low enzyme concentration and to multilayer adsorption at a high enzyme concentration. It may be possible to apply OXDC to degradation of calcium oxalate stones.     

Sibling-size variation in brown trout Salmo trutta in relation to egg size and stream size

Sibling-size variation (SSV), estimated as the coefficient of variation of egg size, was investigated for 13 populations of brown trout Salmo trutta . SSV was negatively correlated with mean egg size both at the population and individual levels. After correction for the effect of mean egg size, SSV was also negatively correlated with stream size. These results provide new information about how salmonid SSV can vary at different ecological scales (individual, population and region). The results are discussed in light of competing theories for explaining SSV: (1) the passive effect hypothesis, stating that egg size variation follows passively from selection on egg size and (2) the bet-hedging hypothesis, stating that high SSV is adaptive in unpredictable environments.     

Cholinergic anti-inflammatory pathway activity and High Mobility Group Box-1 (HMGB1) serum levels in patients with rheumatoid arthritis

High Mobility Group Box-1 (HMGB1) is a cytokine implicated in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. The cholinergic anti-inflammatory pathway, a vagus nerve-dependent mechanism, inhibits HMGB1 release in experimental disease models. Here, we examine the relationship between vagus nerve activity and HMGB1 in patients with RA. We compared RR interval variability, an index of cardiac vagal modulation, HMGB1 and hsCRP serum levels, and disease activity scores in thirteen RA patients and eleven age- and sex-matched controls. In RA patients, serum levels of HMGB1 and hsCRP were elevated as compared with controls (HMGB1=71 ng/mL [45-99] vs. 18 ng/mL [0-40], P<0.0001; hsCRP=14.5 mg/L [0.7-59] vs. 1 mg/L [0.4-2.9], P<0.001). RR interval variability in RA patients was significantly decreased as compared with controls (HF=38 msec2 [14-80] vs. 288 msec2 [38-364], P<0.0001; rMSSD=20.9+/-9.79 msec, 52.6+/-35.3 msec, P<0.01). HMGB1 levels and RR interval variability were significantly related (rho=-0.49, P<0.01). HMGB1 serum levels significantly correlated with disease activity scores (DAS-28) in patients with RA (P=0.004). The study design does not enable a determination of causality, but the results are consistent with the hypothesis that decreased cholinergic anti-inflammatory pathway activity is associated with increased HMGB1 levels in patients with RA.     

Proteomics reveals that redox regulation is disrupted in patients with ethylmalonic encephalopathy

Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide.     

Manual pressure distension of the human saphenous vein changes its biomechanical properties-implication for coronary artery bypass grafting

Patency rates of saphenous vein grafts following coronary artery bypass grafting (CABG) depend on multiple factors. Information regarding the impact of biomechanical properties of vein grafts on patency rates is not available. The objective of the present study was to evaluate whether uncontrolled manual pressure distension during routine preparation of the saphenous vein in CABG-induced changes in the biomechanical properties of the vein. The morphometric and stress-strain properties were studied in isolated segments of the saphenous vein from 12 patients undergoing elective CABG. Six segments were manually distended without pressure control and six were not distended. The mechanical test was performed as a ramp inflation using syringe pump. The vein dimensions were obtained from digitised images at different pressures as well as at the no-load and zero-stress states. The circumferences, the wall and lumen area, the wall thickness, and the outer diameter as function of the applied pressure were largest in the segments with uncontrolled manual distension compared to those without distension (P<0.05). The opening angle and the absolute value of the residual strains were lower (P<0.01) and the circumferential stress-strain curve shifted to the left, indicating the wall became stiffer with uncontrolled manual distension compared to those without distension (P<0.05). In conclusion, manual pressure distension changed the morphometric and biomechanical properties of the saphenous vein. The perspective is that studies on biomechanical properties on the saphenous vein may guide surgeons how to handle graft material without causing major changes of the biomechanical properties during harvesting and preparation.