Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.     

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

Background  

Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells.     

Impacts of Nutrient Reduction on Coastal Communities

Eutrophication due to high anthropogenic nutrient loading has greatly impacted ecological processes in marine coastal waters and, therefore, much effort has been put into reducing nitrogen and phosphorus discharges into European and North-American waters. Nutrient enrichment usually resulted in increase of biomass and production of phytoplankton and microphytobenthos, often coinciding with shifts in species composition within the primary producer community. Consequences of increasing eutrophication for higher trophic levels are still being disputed, and even less is known about the consequences of nutrient reduction on coastal food webs. Here, we present 30-year concurrent field observations on phytoplankton, macrozoobenthos and estuarine birds in the Dutch Wadden Sea, which has been subject to decades of nutrient enrichment and subsequent nutrient reduction. We demonstrate that long-term variations in limiting nutrients (phosphate and silicon) were weakly correlated with biomass and more strongly with community structures of phytoplankton, macrozoobenthos and estuarine birds. Although we cannot conclusively determine if, and if so to what extent, nutrient enrichment and subsequent nutrient reduction actually contributed to the concurrent trends in these communities, it appears likely that part of the variance in the studied coastal communities is related to changes in nutrient loads. Our results imply that nutrient reduction measures should not ignore the potential consequences for policies aimed at bird conservation and exploitation of marine living resources. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ceramide as a TLR4 agonist; a putative signalling intermediate between sphingolipid receptors for microbial ligands and TLR4

Mucosal Toll-like receptors (TLRs) respond to pathogens, but remain inert to the indigenous flora, suggesting that the TLRs can receive pathogen-specific signals. For example, TLR4 signalling is activated in CD14-negative epithelial cells by P-fimbriated, uropathogenic Escherichia coli, but not by lipopolysaccharide. The fimbriae use glycosphingolipids as recognition receptors and there is release of ceramide, which is the membrane-anchoring domain of the receptors. In this study, ceramide was identified as a TLR4 agonist and as a putative signalling intermediate between the glycosphingolipid recognition receptors and TLR4. Exogenous ceramide activated a TLR4-dependent epithelial cell response, as shown by exposing stably transfected TLR4-positive or -negative human embryonal kidney cells to C2 and C6 ceramide. A similar, TLR4-dependent response occurred after deliberate release of endogenous long-chained ceramide with sphingomyelinase. Microbial ligands with glycosphingolipid specificity (P fimbriae or the B subunit of Shiga toxin) were shown to increase the levels of ceramide and to trigger a TLR4-dependent response in epithelial cells. The results show that ceramide activates TLR4 signalling and suggest that this mechanism might allow pathogens to elicit mucosal TLR4 responses by perturbing sphingolipid receptors for virulence ligands like P fimbriae.     

Conquest of the deep,old and cold: an exceptional limpet radiation in Lake Baikal

Lake Baikal is the deepest, oldest and most speciose ancient lake in the world. The lake is characterized by high levels of molluscan species richness and endemicity, including the limpet family Acroloxidae with 25 endemic species. Members of this group generally inhabit the littoral zone, but have been recently found in the abyssal zone at hydrothermal vents and oil-seeps. Here, we use mitochondrial and nuclear data to provide a first molecular phylogeny of the Lake Baikal limpet radiation, and to date the beginning of intra-lacustrine diversification. Divergence time estimates suggest a considerably younger age for the species flock compared with lake age estimates, and the beginning of extensive diversification is possibly related to rapid deepening and cooling during rifting. Phylogenetic relationships and divergence time estimates do not clearly indicate when exactly the abyssal was colonized but suggest a timeframe coincident with the formation of the abyssal in the northern basin (Middle to Late Pleistocene).     

Remarkable heterogeneity in myosin heavy-chain composition of the human young masseter compared with young biceps brachii

Adult human jaw muscles differ from limb and trunk muscles in enzyme-histochemical fibre type composition. Recently, we showed that the human masseter and biceps differ in fibre type pattern already at childhood. The present study explored the myosin heavy-chain (MyHC) expression in the young masseter and biceps muscles by means of gel electrophoresis (GE) and immuno-histochemical (IHC) techniques. Plasticity in MyHC expression during life was evaluated by comparing the results with the previously reported data for adult muscles. In young masseter, GE identified MyHC-I, MyHC-IIa MyHC-IIx and small proportions of MyHC-fetal and MyHC-α cardiac. Western blots confirmed the presence of MyHC-I, MyHC-IIa and MyHC-IIx. IHC revealed in the masseter six isomyosins, MyHC-I, MyHC-IIa, MyHC-IIx, MyHC-fetal, MyHC α-cardiac and a previously not reported isoform, termed MyHC-IIx'. The majority of the masseter fibres co-expressed two to four isoforms. In the young biceps, both GE and IHC identified MyHC-I, MyHC-IIa and MyHC-IIx. MyHC-I predominated in both muscles. Young masseter showed more slow and less-fast and fetal MyHC than the adult and elderly masseter. These results provide evidence that the young masseter muscle is unique in MyHC composition, expressing MyHC-α cardiac and MyHC-fetal isoforms as well as hitherto unrecognized potential spliced isoforms of MyHC-fetal and MyHC-IIx. Differences in masseter MyHC expression between young adult and elderly suggest a shift from childhood to adulthood towards more fast contractile properties. Differences between masseter and biceps are proposed to reflect diverse evolutionary and developmental origins and confirm that the masseter and biceps present separate allotypes of muscle.     

The impact of confounders on the test performance of natriuretic peptides for cardiac dysfunction in subjects aged 80 and older

The hypothesis that natriuretic peptides could be used to identify ‘pancardiac’ damage has been proposed. However, multiple factors are known to influence circulating levels of natriuretic peptides, especially in the very old. Therefore, the impact of confounders on the association between natriuretic peptide levels and cardiac dysfunction was further explored in subjects aged 80 and older. A diagnostic cross-sectional study embedded within the BELFRAIL study (n = 567) was performed. Baseline BNP and NT-proBNP levels were measured and echocardiograms were performed at the subject's home. Cardiac dysfunction was defined as systolic dysfunction, valvular heart disease or isolated severe diastolic dysfunction. Several functional and structural echocardiographic parameters were independently related to circulating levels of natriuretic peptides. Cystatin C, BMI, β blockers, diabetes, heart frequency, usCRP, age and sex were identified as confounders. The prevalence of cardiac dysfunction was 17.1% in the subjects without and 30.8% in the subjects with chronic atrial fibrillation (CAF) or pacemaker (PM). Only in subjects with CAF or PM the C statistic for cardiac dysfunction improved after correcting for confounders. The post-test probability for a negative test (PTP−) ranged from 3.7% to 12.2% and the PTP+ ranged from 21.9% to 62.2% in different strata of confounders. According to these data adjusting for identified confounders does not improve the diagnostic accuracy of the natriuretic peptides for cardiac dysfunction, except in subjects with CAF or PM. Stratifying for individual confounders showed that different cut-off values could be used to optimize the diagnostic characteristics of natriuretic peptides.     

Alternatively folded proteins with unexpected beneficial functions

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.     

Multilevel grouped regression for analyzing self-reported health in relation to environmental factors: the model and its application

A method for modeling the relationship of polychotomous health ratings with predictors such as area characteristics, the distance to a source of environmental contamination, or exposure to environmental pollutants is presented. The model combines elements of grouped regression and multilevel analysis. The statistical model describes the entire response distribution as a function of the predictors so that any measure that summarizes this distribution can be calculated from the model. With the model, polychotomous health ratings can be used, and there is no need for a priori dichotomizing such variables which would lead to loss of information. It is described how, according to the model, various measures describing the response distribution are related to the exposure, and the confidence and tolerance intervals for these relationships are presented. Specific attention is given to the incorporation of random factors in the model. The application that here serves as an example, concerns annoyance from transportation noise. Exposure-response relationships obtained with the described method of modeling are presented for aircraft, road traffic, and railway noise.