Normal mode-based fitting of atomic structure into electron density maps: application to sarcoplasmic reticulum Ca-ATPase

A method for the flexible docking of high-resolution atomic structures into lower resolution densities derived from electron microscopy is presented. The atomic structure is deformed by an iterative process using combinations of normal modes to obtain the best fit of the electron microscopical density. The quality of the computed structures has been evaluated by several techniques borrowed from crystallography. Two atomic structures of the SERCA1 Ca-ATPase corresponding to different conformations were used as a starting point to fit the electron density corresponding to a different conformation. The fitted models have been compared to published models obtained by rigid domain docking, and their relation to the known crystallographic structures are explored by normal mode analysis. We find that only a few number of modes contribute significantly to the transition. The associated motions involve almost exclusively rotation and translation of the cytoplasmic domains as well as displacement of cytoplasmic loops. We suggest that the movements of the cytoplasmic domains are driven by the conformational change that occurs between nonphosphorylated and phosphorylated intermediate, the latter being mimicked by the presence of vanadate at the phosphorylation site in the electron microscopy structure.     

Role of the endothelium on arterial vasomotion

It is well-known that cyclic variations of the vascular diameter, a phenomenon called vasomotion, are induced by synchronous calcium oscillations of smooth muscle cells (SMCs). However, the role of the endothelium on vasomotion is unclear. Some experimental studies claim that the endothelium is necessary for synchronization and vasomotion, whereas others report rhythmic contractions in the absence of an intact endothelium. Moreover, endothelium-derived factors have been shown to abolish vasomotion by desynchronizing the calcium signals in SMCs. By modeling the calcium dynamics of a population of SMCs coupled to a population of endothelial cells, we analyze the effects of an SMC vasoconstrictor stimulation on endothelial cells and the feedback of endothelium-derived factors. Our results show that the endothelium essentially decreases the SMCs calcium level and may move the SMCs from a steady state to an oscillatory domain, and vice versa. In the oscillatory domain, a population of coupled SMCs exhibits synchronous calcium oscillations. Outside the oscillatory domain, the coupled SMCs present only irregular calcium flashings arising from noise modeling stochastic opening of channels. Our findings provide explanations for the published contradictory experimental observations.     

Serial magnetic resonance imaging based assessment of the early effects of an ACE inhibitor on postinfarction left ventricular remodeling in rats

In vivo assessment of treatment efficacy on postinfarct left ventricular (LV) remodeling is crucial for experimental studies. We examined the technical feasibility of serial magnetic resonance imaging (MRI) for monitoring early postinfarct remodeling in rats. MRI studies were performed with a 7-Tesla unit, 1, 3, 8, 15, and 30 days after myocardial infarction (MI) or sham operation, to measure LV mass, volume, and the ejection fraction (EF). Three groups of animals were analyzed: sham-operated rats (n = 6), MI rats receiving lisinopril (n = 11), and MI rats receiving placebo (n = 8). LV dilation occurred on day 3 in both MI groups. LV end-systolic and end-diastolic volumes were significantly lower in lisinopril-treated rats than in placebo-treated rats at days 15 and 30. EF was lower in both MI groups than in the sham group at all time points, and did not differ between the MI groups during follow-up. Less LV hypertrophy was observed in rats receiving lisinopril than in rats receiving placebo at days 15 and 30. We found acceptable within- and between-observer agreement and an excellent correlation between MRI and ex vivo LV mass (r = 0.96; p < 0.001). We demonstrated the ability of MRI to detect the early beneficial impact of angiotensin-converting enzyme (ACE) inhibitors on LV remodeling. Accurate and noninvasive, MRI is the tool of choice to document response to treatment targeting postinfarction LV remodeling in rats.     

P-deficiency increases the O2 uptake per N2 reduced in alfalfa

Nodulated alfalfa (Medicago sativa L. cv. Saranac) plants were grown in hydroponics at P-sufficient and P-deficient supply levels. After 5 weeks of growth, dry matter accumulation, nodulation, total N and P accumulation, as well as 15N2 uptake, were measured. Moreover, the response of nodule O2-uptake to raising external pO2 was determined in an open-flow measurement system and nodule permeability was calculated. Plants in the P-deficient supply treatment had a lower P concentration in all organs. In both treatments the highest P concentration was found in nodules. In the P-deficient supply treatment plants formed less dry matter, had a lower shoot/root ratio, less nodulation, decreased total N accumulation, and lower 15N2 uptake per dry matter nodule. Nodules in the P-deficient treatment were, on average, smaller and had a higher O2 uptake per N2 reduced, coinciding with increased nodule permeability and conductance. Thus increased oxygen uptake appears to be a mechanism to adjust nodule metabolism to P deficiency in indeterminate N2-fixing nodules such as in alfalfa, as has previously been shown for determinate nodule forms.     

Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.     

A Pressurized Nitrogen Counterbalance to Cortical Glutamatergic Pathway Stimulation

Previous microdialysis studies performed in rats have revealed a decrease of striatal dopamine and glutamate induced by nitrogen narcosis. We sought to establish the hypothetical role of the glutamatergic corticostriatal pathway because of the glutamate deficiency which occurs in the basal ganglia in this hyperbaric syndrome. Retrodialysis with 1 mM of Saclofen and 100 mM of KCl in the prefrontal cortex under normobaric conditions led to an increase in striatal levels of glutamate by 95.2% and no changes in dopamine levels. Under 3 MPa of nitrogen and with the infusion, the rate of striatal glutamate decreased by 51.3%, to a greater extent than under pressurised nitrogen alone (−23.8%). The rate of dopamine decreased, which also occurred under pressurised nitrogen (−36.9 and −31.4%, respectively). In conclusion, the function of the corticostriatal pathway is affected by nitrogen under pressure. This suggests that the nitrogen-induced break point seems to be located at the glutamatergic striatopetal neurons.     

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.