Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization,efficient signaling,and interaction with β-arrestin 2

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and β-arrestin (β-arr 319–418) and by means of cell lines that expressed various endogenous levels of β-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and β-arrestin and even more profoundly in cells lacking both β-arrestin1 and β-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with β-arrestin2. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the Slovenian Ministry of Higher Education, Science, and Technology (grant number 0406-007) and a Slovenian-Danish collaboration grant (BI-DK/06-07-007).     

Fitness level of adult economically active population in the Republic of Croatia estimated by EUROFIT system

The study presents the results of the EUROFIT CROATIA project that was conducted according to the instructions of Council of Europe. The aim of the study was evaluation of fitness level of the employed Croatian population. The measurements comprised EUROFIT battery of tests as well as Baecke's questionnaire for evaluation of habitual physical activity. The sample was comprised of 1628 active inhabitants, 18-60 years of age, all of who were employed and living in Croatia. The obtained results of functional, motor and morphological characteristics were presented according to the sex and age. The results shower poor aerobic capacity, high obesity indicators and low motor performance in both sexes, which pointed to the increased risk for developing many cardiovascular or musculo-skeletal diseases. The overall fitness level of Croatian population showed to be unsatisfactory and it should be improved by engagement in sports-recreational activities.     

The -429 T/C and -374 T/A gene polymorphisms of the receptor of advanced glycation end products gene (RAGE) are not risk factors for coronary artery disease in Slovene population with type 2 diabetes

Receptor for advanced glycation end products (RAGE) plays a role in atherosclerosis in diabetics. There are two functional polymorphisms in the promoter of the RAGE gene (-429T/C and -374T/A). The aim of this study was to look for a relationship between the -429T/C and the -374T/A gene polymorphisms of the RAGE gene and the development of coronary artery disease (CAD) in the Slovene population with type 2 diabetes of duration longer than 10 years. One hundred and sixty-eight subjects with diabetes and CAD were compared to 241 diabetic subjects without CAD. The -429T/C and the -374T/A RAGE genotype distributions in patients with CAD (-429T/C: CC: 3%, TC: 31%, TT: 66.0%; -374T/A:AA: 7.7%, TA: 48.2%, TT: 44.1%) were not significantly different from those in patients without CAD (-429 T/C: CC: 1.7%, TC: 26.1%, TT: 72.2%; -374T/A: AA: 11.2%, TA: 43.2%, TT: 45.6%). Our study failed to demonstrate an association between either the -429T/C or the -374T/A gene polymorphism of the RAGE gene and CAD in the Slovene population with type 2 diabetes of duration longer than 10 years.     

Overweight and obesity in Slovenian schoolgirls, 1991-2006

The proportion of overweight children and adults has been growing rapidly in the last few years in many European and other countries. Certain consequences of overweight are already manifested in youth; in adulthood they are one of the main causes of death and several diseases. The study examined the proportion of overweight and obese Slovenian girls aged 7 to 18 with the use of an annually repeated cross-sectional study. The study lasted from 1991 to 2006 and was based on the body mass index according to IOTF norms. The results show that in this period the proportion of overweight girls increased by almost 30% (from 13.5% to 18.8%), whereas the proportion of obese girls doubled (from 2.3% to 4.6%). The prevalence of overweight and obesity is highest in childhood and early adolescence where it is around two to three times higher than at the age of 18.     

Physical fitness, menstrual cycle disorders and smoking habit in Croatian National Ballet and National Folk Dance Ensembles

The study investigated differences in morphological, motor and functional abilities between folk and ballet dancers. The sample comprised 51 female subjects: Croatian National Ballet (N=30) and Croatian National Folk Ensemble "LADO" (N=21). The data regarding menstrual cycle, menarche, number of births and smoking habit were collected and the morphological, motor and functional abilities measured. Significant correlations between the amount of fat tissue and number of births were found in both groups. Folk dancers were as tall as ballet dancers but weighted more and had a larger body frame (p<0.001). Ballet dancers were more flexible but there were no differences in absolute maximal oxygen uptake (2.65 vs. 2.35 L/min, p=0.101). Still, as the ballet dancers weighted less, their relative maximal oxygen uptake was significantly higher (37.62 vs. 50.22 mL/kg/min, p<0.001). Also, a high number of 45% of smokers among professional ballet and professional folk dancers was found.     

An ESR characterization of micelles and vesicles formed in aqueous decanoic acid/sodium decanoate systems using different spin labels

Aqueous decanoic acid/sodium decanaote systems were studied as a function of pH and concentration, up to 0.3 M decanoic acid/sodium decanoate, by electron spin resonance (ESR) spectroscopy using three different amphiphilic spin labels. The distribution of the spin labels between vesicles and micelles as well as their dynamic properties were determined by quantitative analysis of the ESR spectra using two novel simulation software packages. Rotational correlation time of the labels in micelles was found to increase with decreasing pH, with substantial increase in the region where vesicles were formed (7.8相似文献   

Arrhythmogenic right ventricular dysplasia and sudden cardiac death in Croatians' young athletes in 25 years

The paper deals with the sudden cardiac death during training in male athletes in Croatia. The data are a part of a retrospective study dealing with 67 sudden death due to physical activity in men in Croatia during 25 years: from January 1, 1986 to December 31, 2010. Two of them suddenly died during training due to malignant ventricular arrhythmia because of the arrhythmogenic right ventricular dysplasia. First was a short trails runner aged 24, with no any previous physical discomforts, who suddenly collapsed and died during training. The second was a soccer player aged 13, with no any previous physical discomfort, who suddenly collapsed and died during training. A sudden cardiac death due to physical exercise in young athletes in Croatia suffered of arrhythmogenic right ventricular dysplasia reached 0.07/ 100.000 yearly (p = 0.00000), in all young athletes suffered of heart diseases reached 0.19/100 000 (p = 0.00005), and in the total male population aged 15-40 engaged in sports and recreational physical exercise: 0.71/100.0000 (p = 0.00001).     

HaloTag-based purification of functional human kinases from mammalian cells

Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.     

A transgenic mouse line expressing cre recombinase in pancreatic β-cells

Transgenic mouse lines expressing Cre recombinase in a cell-specific and tissue-specific manner are essential tools for studying gene function and for developing suitable models for human diseases. Here, we used an expression cassette containing the full 5' untranslated region of the porcine insulin gene to generate a mouse line expressing Cre recombinase specifically in pancreatic β-cells by pronuclear DNA microinjection. We obtained a founder animal that transmitted the construct to its descendants in a Mendelian fashion and whose descendants showed a clear activation of β-galactosidase expression in pancreatic β-cells after crossing into the ROSA26 lacZ reporter mouse line. Cre expression in other organs was negative except for the kidney, intestine, and the cerebral pons where β-galactosidase activity was detected in a small percentage of the cells. This new mouse line is a valuable tool for recombination of floxed alleles in pancreatic β-cells in vivo.