Polarisation, key to good localisation

Polarisation of cells is crucial for vectorial transport of ions and solutes. In literature, however, proteins specifically targeted to the apical or basolateral membrane are often studied in non-polarised cells. To investigate whether these data can be extrapolated to expression in polarised cells, we studied several membrane-specific proteins. In polarised MDCK cells, the Aquaporin-2 water channel resides in intracellular vesicles and apical membrane, while the vasopressin-type 2 receptor, anion-exchanger 1 (AE1) protein and E-Cadherin mainly localise to the basolateral membrane. In non-polarised MDCK cells, however, Aquaporin-2 localises, besides plasma membrane, mainly in the Golgi complex, while the others show a dispersed staining throughout the cell. Moreover, while AQP2 mutants in dominant nephrogenic diabetes insipidus are missorted to different organelles in polarised cells, they all predominantly localise to the Golgi complex in non-polarised MDCK cells. Additionally, the maturation of V2R, and likely its missorting, is affected in transiently-transfected compared to stably-transfected cells. In conclusion, we show that the use of stably-transfected polarised cells is crucial in interpreting the processing and the localisation of membrane targeted proteins.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Environmental and morphological factors influencing predatory behaviour by invasive non-indigenous gammaridean species

Predatory behaviour seems to be more frequent in invasive gammaridean species than in native ones. This results in the exclusion of other, mostly native gammaridean species and a change in benthic communities. The present study analysed the influence of environmental factors (water temperature) and morphological factors (sex, body parts involved in catching and holding prey) on the predatory behaviour of Dikerogammarus villosus. A diet study of invasive relatives of D. villosus showed that predation intensity is especially high in spring and summer, that is, at increasing and high temperatures. Experiments with D. villosus in climate rooms at various temperatures, using the native Gammarus fossarum as prey, showed that the average predation rate by both sexes gradually increased over the temperature range from 5 to 30°C. Natural mortality during the experiments was negligible compared to losses due to predation. At each temperature, the predation rate by females was lower than that by males. Males showed a steep allometric growth of body parts involved in the process of catching and holding prey, compared to females at increasing body size in a number of measurements. This may explain the difference in predatory behaviour between males and females, which plays a role in intraguild predation a supposed mechanism for species displacement.     

Development of a sensitive and selective assay for the determination of procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) in plasma

To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.     

Improving guideline adherence in the treatment of atrial fibrillation by implementing an integrated chronic care program

Background / Objectives. Atrial fibrillation (AF) is a very frequent and complex disease often associated with other medical conditions. The Euro Heart Survey (EHS) on AF showed that adherence to guidelines may reduce morbidity and mortality in AF patients. Therefore a nurse-driven, guideline-based, ICT-supported integrated chronic care program (ICCP) was developed and implemented in daily practice. The objective of this study is to evaluate the clinical feasibility of the ICCP, with guideline adherence as the endpoint. Methods. 111 ambulant patients referred for treatment of their AF were enrolled in the ICCP. In this group, patients underwent standardised clinical testing and were subsequently managed by a nurse, supported by a dedicated ICT program and supervised by cardiologists. For comparison, we used a recent historical control group of 102 patients who participated in the Maastricht part of the Euro Heart Survey (EHS) on AF. Results. Guideline adherence was excellent within the ICCP and compared favourably with the EHS-AF data concerning both clinical testing (trigger factors recorded in 100 vs. 44%; echocardiogram performed in 99 vs. 88%; thyroid-stimulating hormone level recorded in 96% vs. 63%) as well as treatment (antithrombotic therapy in 90 vs. 78%; rhythm control avoided in completely asymptomatic patients in 100 vs. 54%; class I drugs avoided in patients with structural heart disease in 90 vs. 95%; rhythm control avoided in permanent AF patients in 100 vs. 92%). Conclusion. The high level of guideline adherence suggests that a nurse-driven, guideline-based, ICT-supported ICCP for AF patients is feasible. (Neth Heart J 2010;18:471-7.)     

Fasting ghrelin does not predict food intake after short-term energy restriction

Objective: To study the role of ghrelin as a hunger signal during energy restriction and to test the hypothesis that changes in fasting leptin concentrations during energy restriction are associated with changes in fasting ghrelin concentrations. Research Methods and Procedures: Thirty‐five healthy, lean men (23 ± 3 years of age; BMI: 22.3 ± 1.6 kg/m²) participated in a controlled intervention study. Fasting ghrelin and leptin concentrations were measured before and after 2 days of 62% energy restriction and after a 2‐day period of ad libitum food intake. Energy intake during the latter period was assessed. Results: On average, ghrelin concentrations did not change (0.05 μg/liter; 95% confidence interval, ?0.03; 0.12) during energy restriction. Changes in ghrelin concentration during energy restriction were not associated with energy intake during the ad libitum period (r = 0.07; not significant). Ad libitum energy intake was, however, associated with the change in ghrelin concentrations during the same period (r = ?0.34; p = 0.05). Ghrelin and leptin concentrations were not associated. In addition, the ratio of percentage changes in ghrelin and leptin during energy restriction was not correlated with ad libitum food intake after energy restriction (r = ?0.26; p = 0.14). Discussion: Fasting ghrelin concentrations did not rise after a 2‐day energy restriction regimen. Moreover, changes in ghrelin concentrations during energy restriction were not associated with subsequent ad libitum food intake, suggesting that fasting ghrelin does not act as a hunger signal to the brain. The data did not support our hypothesis that leptin suppresses ghrelin levels.     

PDZ domains – glue and guide

PDZ domains are small globular building blocks that are amongst the most abundant protein interaction domains in organisms. Over the past several years an avalanche of data has implicated these modules in the clustering, targeting and routing of associating proteins. An overview is given of the types of interactions displayed by PDZ domains and how this relates to the current knowledge on their spatial structure. Furthermore, the different levels on which PDZ – ligand binding can be regulated and the consequences of PDZ domain-mediated clustering for activity, routing and targeting of interacting proteins will be addressed. Finally, some cell and animal models that illustrate the impact of PDZ domain-containing proteins on (multi-) cellular processes will be discussed.     

Linkage analysis of a derived glucose phenotype in the Genetic Analysis Workshop 13 simulated data using a variety of Haseman-Elston based regression methods

A variety of Haseman-Elston type regression procedures were used to perform a genome scan across five chromosomes, using replicates 1-5 of the Genetic Analysis Workshop 13 simulated data. The traits of interest were variables corresponding to 'baseline' and 'slope' effects derived from the fasting glucose phenotypes. Performance in terms of detecting the locations of known trait loci was poor for all methods, even when all five replicates were combined to produce a large data set (9230 sib pairs). All methods performed well, however, when applied to new simulated data in which the true genetic effects were allowed to explain a greater proportion of the overall variance.